Processes for producing fermentation products

ABSTRACT

The present invention relates to processes for producing fermentation products from starch-containing material, wherein an alpha-amylase and optionally a thermostable protease, pullulanase and/or glucoamylase are present and/or added during liquefaction, wherein a cellulolytic composition is present and/or added during fermentation or simultaneous saccharification and fermentation. The invention also relates to a composition suitable for use in a process of the invention.

FIELD OF THE INVENTION

The present invention relates to processes for producing fermentationproducts from starch-containing material. The invention also relates toa composition suitable for use in a process of the invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Production of fermentation products, such as ethanol, fromstarch-containing material is well-known in the art. Industrially twodifferent kinds of processes are used today. The most commonly usedprocess, often referred to as a “conventional process”, and includesliquefying gelatinized starch at high temperature using typically abacterial alpha-amylase, followed by simultaneous saccharification andfermentation carried out in the presence of a glucoamylase and afermentation organism. Another well-known process, often referred to asa “raw starch hydrolysis”-process (RSH process), includes simultaneouslysaccharifying and fermenting granular starch below the initialgelatization temperature typically in the presence of at least aglucoamylase.

Despite significant improvement of fermentation product productionprocesses over the past decade a significant amount of residual starchmaterial is not converted into the desired fermentation product, such asethanol. At least some of the unconverted residual starch material,e.g., sugars and dextrins, is in the form of non-fermentable Maillardproducts.

Therefore, there is still a desire and need for providing processes forproducing fermentation products, such as ethanol, from starch-containingmaterial that can provide a higher fermentation product yield, or otheradvantages, compared to a conventional process.

SUMMARY OF THE INVENTION

The present invention relates to processes of producing fermentationproducts, such as ethanol, from starch-containing material using afermenting organism.

In the first aspect the invention relates to processes for producingfermentation products, such as ethanol, from starch-containing materialcomprising the steps of:

i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.; and    -   optionally a carbohydrate-source generating enzyme;

ii) saccharifying using a carbohydrate-source generating enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present or added duringfermentation or simultaneous saccharification and fermentation.

Suitable cellulolytic compositions are described below. In a preferredembodiment the cellulolytic composition is derived from Trichodermareesei.

In a preferred embodiment liquefaction is carried out at a temperaturebetween from 70-100° C., such as between 75-95° C., such as between75-90° C., preferably between 80-90° C., such as 82-88° C., such asaround 85° C.

In an embodiment the pH during liquefaction is from 4.5-5.0, such asbetween 4.5-4.8. In another embodiment liquefaction is carried out at apH above 5.0-6.5, such as above 5.0-6.0, such as above 5.0-5.5, such asbetween 5.2-6.2, such as around 5.2, such as around 5.4, such as around5.6, such as around 5.8.

In a second aspect the invention relates to an enzyme compositioncomprising:

-   -   an alpha-amylase;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.; and    -   optionally a pullulanase;    -   optionally a carbohydrate-source generating enzyme.

The alpha-amylase present may be any alpha-amylase, preferably abacterial alpha-amylase, in particular from Bacillus stearothermophilus,especially a thermostable variant thereof. Examples of thermostablevariants are given below. Preferred examples include alpha-amylasesselected from the group of Bacillus stearothermophilus alpha-amylasevariants:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   I181*+G182*+N193F+V59A+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S.

The composition of the invention optionally comprises a pullulanase. Thepullulanase may be a family GH57 pullulanase, such as a pullulanasewhich includes an X47 domain as disclosed in WO 2011/087836. Moreexamples are given in the “Pullulanase Present and/or Added DuringLiquefaction”-section below.

In embodiments of the invention a thermostable protease and/or acarbohydrate-source generating enzyme, in particular a glucoamylases,are optionally present.

Examples of thermostable proteases can be found in the “Protease Presentand/or Added During Liquefaction”-section below. In a preferredembodiment the thermostable protease is a variant of the Thermoascusaurantiacus protease shown in SEQ ID NO: 3 herein or a protease derivedfrom a strain of Pyrococcus furiosus, in particular the one shown in SEQID NO: 13 herein, SEQ ID NO: 29 herein or disclosed in U.S. Pat. No.6,358,726-B1.

Examples of suitable optional carbohydrate-source generating enzymes,preferably thermostable carbohydrate-source generating enzymes, inparticular a thermostable glucoamylase, can be found in the“Carbohydrate-Source Generating Enzymes Present and/or Added DuringLiquefaction”-section below.

In an embodiment the carbohydrate-source generating enzyme, inparticular a glucoamylase, is Penicillium oxalicum glucoamylase, or avariant thereof.

Other enzyme activities may also be present.

DEFINITIONS Enzymes:

Cellulolytic composition, cellulolytic enzymes or cellulase means apreparation comprising one or more (e.g., several) enzymes thathydrolyze a cellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

Cellulolytic enzyme activity is determined by measuring the increase inhydrolysis of a cellulosic material by cellulolytic enzyme(s) under thefollowing conditions: 1-50 mg of cellulolytic enzyme protein/g ofcellulose in Pretreated Corn Stover (“PCS”) (or other pretreatedcellulosic material) for 3-7 days at a suitable temperature, e.g., 50°C., 55° C., or 60° C., compared to a control hydrolysis without additionof cellulolytic enzyme protein. Typical conditions are 1 ml reactions,washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5,1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours, sugar analysis byAMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA).

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at a suitable temperature, e.g., 50° C., 55° C., or 60° C., andpH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS). In an aspect, a mixture ofCELLUCLAST® 1.5 L (Novozymes NS, Bagsværd, Denmark) in the presence of2-3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The GH61 polypeptide having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.

For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20 (polyoxyethylene sorbitanmonolaurate).

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178).

Cellobiohydrolase activity is determined according to the proceduresdescribed by Lever et al., 1972, Anal. Biochem. 47: 273-279; vanTilbeurgh et al., 1982, FEBS Letters, 149: 152-156; van Tilbeurgh andClaeyssens, 1985, FEBS Letters, 187: 283-288; and Tomme et al., 1988,Eur. J. Biochem. 170: 575-581. In the present invention, the Tomme etal. method can be used to determine cellobiohydrolase activity.

Endoglucanase: The term “endoglucanase” means an endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzesendohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulosederivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components.

Endoglucanase activity can be determined by measuring reduction insubstrate viscosity or increase in reducing ends determined by areducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24:452-481). For purposes of the present invention, endoglucanase activityis determined using carboxymethyl cellulose (CMC) as substrate accordingto the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH5, 40° C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to processes of producing fermentationproducts, such as ethanol from starch-containing material using afermenting organism.

In the first aspect the invention relates to processes for producingfermentation products, preferably ethanol, comprising the steps of:

i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.; and    -   optionally a carbohydrate-source generating enzyme;

ii) saccharifying using a carbohydrate-source generating enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present or added duringfermentation or simultaneous saccharification and fermentation.

Steps ii) and iii) are carried out either sequentially orsimultaneously. In a preferred embodiment steps ii) and iii) are carriedout simultaneously. The alpha-amylase, optional thermostable protease,optional carbohydrate-source generating enzyme, preferably glucoamylase,and/or, optional a pullulanase, may be added before and/or duringliquefaction step i). A composition of the invention may suitably beused in a process of the invention. However, the enzymes may also beadded separately. Examples of alpha-amylases can be found in the“Alpha-Amylase Present and/or Added During Liquefaction”-section below.Examples of thermostable proteases can be found in the “Protease Presentand/or Added During Liquefaction”-section below. Examples of suitableoptional carbohydrate-source generating enzymes, preferably thermostablecarbohydrate-source generating enzymes, in particular a thermostableglucoamylase, can be found in the “Carbohydrate-Source GeneratingEnzymes Present and/or Added During Liquefaction”-section below. Asuitable optional pullulanase can be found in the “Pullulanase Presentand/or Added During Liquefaction”-section below.

The pH during liquefaction may be between 4-7. In an embodiment the pHduring liquefaction is from 4.5-5.0, such as between 4.5-4.8. In anotherembodiment liquefaction is carried out at a pH above 5.0-6.5, such asabove 5.0-6.0, such as above 5.0-5.5, such as between 5.2-6.2, such asaround 5.2, such as around 5.4, such as around 5.6, such as around 5.8.

According to the invention the temperature is above the initialgelatinization temperature. The term “initial gelatinizationtemperature” refers to the lowest temperature at which solubilization ofstarch, typically by heating, begins. The temperature can vary fordifferent starches.

In an embodiment the temperature during liquefaction step i) is in therange from 70-100° C., such as between 75-95° C., such as between 75-90°C., preferably between 80-90° C., such as between 82-88° C., such asaround 85° C.

In an embodiment, the process of the invention further comprises, priorto the step i), the steps of:

a) reducing the particle size of the starch-containing material,preferably by dry milling;

b) forming a slurry comprising the starch-containing material and water.

The starch-containing starting material, such as whole grains, may bereduced in particle size, e.g., by milling, in order to open up thestructure, to increase surface area, and allowing for furtherprocessing. Generally there are two types of processes: wet and drymilling. In dry milling whole kernels are milled and used. Wet millinggives a good separation of germ and meal (starch granules and protein).Wet milling is often applied at locations where the starch hydrolysateis used in production of, e.g., syrups. Both dry and wet milling arewell known in the art of starch processing. According to the presentinvention dry milling is preferred. In an embodiment the particle sizeis reduced to between 0.05 to 3.0 mm, preferably 0.1-0.5 mm, or so thatat least 30%, preferably at least 50%, more preferably at least 70%,even more preferably at least 90% of the starch-containing material fitthrough a sieve with a 0.05 to 3.0 mm screen, preferably 0.1-0.5 mmscreen. In another embodiment at least 50%, preferably at least 70%,more preferably at least 80%, especially at least 90% of thestarch-containing material fit through a sieve with #6 screen.

The aqueous slurry may contain from 10-55 w/w-% dry solids (DS),preferably 25-45 w/w-% dry solids (DS), more preferably 30-40 w/w-% drysolids (DS) of starch-containing material.

The slurry may be heated to above the initial gelatinizationtemperature, preferably to between 80-90° C., between pH 4-7, preferablybetween 4.5-5.0 or 5.0 and 6.0, for 30 minutes to 5 hours, such asaround 2 hours.

The alpha-amylase, optional thermostable protease, optionalcarbohydrate-source generating enzyme, in particular thermostableglucoamylase, and/or optional pullulanase may initially be added to theaqueous slurry to initiate liquefaction (thinning). In an embodimentonly a portion of the enzymes is added to the aqueous slurry, while therest of the enzymes are added during liquefaction step i).

Liquefaction step i) is according to the invention carried out for 0.5-5hours, such as 1-3 hours, such as typically around 2 hours.

The aqueous slurry may in an embodiment be jet-cooked to furthergelatinize the slurry before being subjected to liquefaction in step i).The jet-cooking may be carried out at a temperature between 110-145° C.,preferably 120-140° C., such as 125-135° C., preferably around 130° C.for about 1-15 minutes, preferably for about 3-10 minutes, especiallyaround about 5 minutes.

Saccharification and Fermentation

One or more carbohydrate-source generating enzymes, in particularglucoamylase, may be present and/or added during saccharification stepii) and/or fermentation step iii). The carbohydrate-source generatingenzyme may preferably be a glucoamylase, but may also be an enzymeselected from the group consisting of: beta-amylase, maltogenic amylaseand alpha-glucosidase. The carbohydrate-source generating enzyme addedduring saccharification step ii) and/or fermentation step iii) istypically different from the optional carbohydrate-source generatingenzyme, in particular thermostable glucoamylase, optionally added duringliquefaction step i). In a preferred embodiment the carbohydrate-sourcegenerating enzymes, in particular glucoamylase, is added together with afungal alpha-amylase. Examples of carbohydrate-source generatingenzymes, including glucoamylases, can be found in the“Carbohydrate-Source Generating Enzyme Present and/or Added DuringSaccharification and/or Fermentation”-section below.

When doing sequential saccharification and fermentation,saccharification step ii) may be carried out at conditions well-known inthe art. For instance, the saccharification step ii) may last up to fromabout 24 to about 72 hours. In an embodiment pre-saccharification isdone. Pre-saccharification is typically done for 40-90 minutes at atemperature between 30-65° C., typically about 60° C.Pre-saccharification is in an embodiment followed by saccharificationduring fermentation in simultaneous saccharification and fermentation(“SSF). Saccharification is typically carried out at temperatures from20-75° C., preferably from 40-70° C., typically around 60° C., and at apH between 4 and 5, normally at about pH 4.5.

Simultaneous saccharification and fermentation (“SSF”) is widely used inindustrial scale fermentation product production processes, especiallyethanol production processes. When doing SSF the saccharification stepii) and the fermentation step iii) are carried out simultaneously. Thereis no holding stage for the saccharification, meaning that a fermentingorganism, such as yeast, and enzyme(s), may be added together. However,it is also contemplated to add the fermenting organism and enzyme(s)separately. SSF is according to the invention typically carried out at atemperature from 25° C. to 40° C., such as from 28° C. to 35° C., suchas from 30° C. to 34° C., preferably around about 32° C. In anembodiment fermentation is ongoing for 6 to 120 hours, in particular 24to 96 hours. In an embodiment the pH is between 3.5-5, in particularbetween 3.8 and 4.3.

Fermentation Medium

“Fermentation media” or “fermentation medium” refers to the environmentin which fermentation is carried out. The fermentation medium includesthe fermentation substrate, that is, the carbohydrate source that ismetabolized by the fermenting organism. According to the invention thefermentation medium may comprise nutrients and growth stimulator(s) forthe fermenting organism(s). Nutrient and growth stimulators are widelyused in the art of fermentation and include nitrogen sources, such asammonia; urea, vitamins and minerals, or combinations thereof.

Fermenting Organisms

The term “Fermenting organism” refers to any organism, includingbacterial and fungal organisms, especially yeast, suitable for use in afermentation process and capable of producing the desired fermentationproduct. Especially suitable fermenting organisms are able to ferment,i.e., convert, sugars, such as glucose or maltose, directly orindirectly into the desired fermentation product, such as ethanol.Examples of fermenting organisms include fungal organisms, such asyeast. Preferred yeast includes strains of Saccharomyces spp., inparticular, Saccharomyces cerevisiae.

Suitable concentrations of the viable fermenting organism duringfermentation, such as SSF, are well known in the art or can easily bedetermined by the skilled person in the art. In one embodiment thefermenting organism, such as ethanol fermenting yeast, (e.g.,Saccharomyces cerevisiae) is added to the fermentation medium so thatthe viable fermenting organism, such as yeast, count per mL offermentation medium is in the range from 10⁵ to 10¹², preferably from10⁷ to 10¹⁰, especially about 5×10⁷.

Examples of commercially available yeast includes, e.g., RED START™ andETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI(available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL(available from DSM Specialties).

Starch-Containing Materials

Any suitable starch-containing material may be used according to thepresent invention. The starting material is generally selected based onthe desired fermentation product. Examples of starch-containingmaterials, suitable for use in a process of the invention, include wholegrains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum,rice, peas, beans, or sweet potatoes, or mixtures thereof or starchesderived there from, or cereals. Contemplated are also waxy and non-waxytypes of corn and barley. In a preferred embodiment thestarch-containing material, used for ethanol production according to theinvention, is corn or wheat.

Fermentation Products

The term “fermentation product” means a product produced by a processincluding a fermentation step using a fermenting organism. Fermentationproducts contemplated according to the invention include alcohols (e.g.,ethanol, methanol, butanol; polyols such as glycerol, sorbitol andinositol); organic acids (e.g., citric acid, acetic acid, itaconic acid,lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone);amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂); antibiotics(e.g., penicillin and tetracycline); enzymes; vitamins (e.g.,riboflavin, B₁₂, beta-carotene); and hormones. In a preferred embodimentthe fermentation product is ethanol, e.g., fuel ethanol; drinkingethanol, i.e., potable neutral spirits; or industrial ethanol orproducts used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry andtobacco industry. Preferred beer types comprise ales, stouts, porters,lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcoholbeer, low-calorie beer or light beer. Preferably processes of theinvention are used for producing an alcohol, such as ethanol. Thefermentation product, such as ethanol, obtained according to theinvention, may be used as fuel, which is typically blended withgasoline. However, in the case of ethanol it may also be used as potableethanol.

Recovery

Subsequent to fermentation, or SSF, the fermentation product may beseparated from the fermentation medium. The slurry may be distilled toextract the desired fermentation product (e.g., ethanol). Alternativelythe desired fermentation product may be extracted from the fermentationmedium by micro or membrane filtration techniques. The fermentationproduct may also be recovered by stripping or other method well known inthe art.

Alpha-Amylase Present and/or Added During Liquefaction

According to the invention an alpha-amylase is present and/or addedduring liquefaction together with an optional thermostable protease,optional carbohydrate-source generating enzyme, in particular athermostable glucoamylase, and/or optional pullulanase.

The alpha-amylase added during liquefaction step i) may be anyalpha-amylase. Preferred are bacterial alpha-amylases, which typicallyare stable at temperature used during liquefaction.

Bacterial Alpha-Amylase

The term “bacterial alpha-amylase” means any bacterial alpha-amylaseclassified under EC 3.2.1.1. A bacterial alpha-amylase used according tothe invention may, e.g., be derived from a strain of the genus Bacillus,which is sometimes also referred to as the genus Geobacillus. In anembodiment the Bacillus alpha-amylase is derived from a strain ofBacillus amyloliquefaciens, Bacillus licheniformis, Bacillusstearothermophilus, or Bacillus subtilis, but may also be derived fromother Bacillus sp.

Specific examples of bacterial alpha-amylases include the Bacillusstearothermophilus alpha-amylase of SEQ ID NO: 3 in WO 99/19467, theBacillus amyloliquefaciens alpha-amylase of SEQ ID NO: 5 in WO 99/19467,and the Bacillus licheniformis alpha-amylase of SEQ ID NO: 4 in WO99/19467 (all sequences are hereby incorporated by reference). In anembodiment the alpha-amylase may be an enzyme having a degree ofidentity of at least 60%, e.g., at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% to any of the sequences shown in SEQ ID NOS: 3, 4 or 5,respectively, in WO 99/19467.

In an embodiment the alpha-amylase may be an enzyme having a degree ofidentity of at least 60%, e.g., at least 70%, at least 80%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% to any ofthe sequences shown in SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1herein.

In a preferred embodiment the alpha-amylase is derived from Bacillusstearothermophilus. The Bacillus stearothermophilus alpha-amylase may bea mature wild-type or a mature variant thereof. The mature Bacillusstearothermophilus alpha-amylases may naturally be truncated duringrecombinant production. For instance, the Bacillus stearothermophilusalpha-amylase may be a truncated so it has around 491 amino acids(compared to SEQ ID NO: 3 in WO 99/19467.

The Bacillus alpha-amylase may also be a variant and/or hybrid. Examplesof such a variant can be found in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents arehereby incorporated by reference). Specific alpha-amylase variants aredisclosed in U.S. Pat. Nos. 6,093,562, 6,187,576, 6,297,038, and7,713,723 (hereby incorporated by reference) and include Bacillusstearothermophilus alpha-amylase (often referred to as BSGalpha-amylase) variants having a deletion of one or two amino acids atpositions R179, G180, I181 and/or G182, preferably a double deletiondisclosed in WO 96/23873—see, e.g., page 20, lines 1-10 (herebyincorporated by reference), preferably corresponding to deletion ofpositions I181 and G182 compared to the amino acid sequence of Bacillusstearothermophilus alpha-amylase set forth in SEQ ID NO: 3 disclosed inWO 99/19467 or SEQ ID NO: 1 herein or the deletion of amino acids R179and G180 using SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1 herein fornumbering (which reference is hereby incorporated by reference). Evenmore preferred are Bacillus alpha-amylases, especially Bacillusstearothermophilus alpha-amylases, which have a double deletioncorresponding to a deletion of positions 181 and 182 and furthercomprise a N193F substitution (also denoted I181*+G182*+N193F) comparedto the wild-type BSG alpha-amylase amino acid sequence set forth in SEQID NO: 3 disclosed in WO 99/19467 or SEQ ID NO: 1 herein. The bacterialalpha-amylase may also have a substitution in a position correspondingto S239 in the Bacillus licheniformis alpha-amylase shown in SEQ ID NO:4 in WO 99/19467, or a S242 variant of the Bacillus stearothermophilusalpha-amylase of SEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1 herein.

In an embodiment the variant is a S242A, E or Q variant, preferably aS242Q variant, of the Bacillus stearothermophilus alpha-amylase (usingSEQ ID NO: 1 herein for numbering).

In an embodiment the variant is a position E188 variant, preferablyE188P variant of the Bacillus stearothermophilus alpha-amylase (usingSEQ ID NO: 1 herein for numbering).

The bacterial alpha-amylase may in an embodiment be a truncated Bacilluslicheniformis alpha-amylase. Especially the truncation is so that theBacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 in WO99/19467 or SEQ ID NO: 1 herein, is around 491 amino acids long, such asfrom 480 to 495 amino acids long.

Bacterial Hybrid Alpha-Amylases

The bacterial alpha-amylase may also be a hybrid bacterialalpha-amylase, e.g., an alpha-amylase comprising 445 C-terminal aminoacid residues of the Bacillus licheniformis alpha-amylase (shown in SEQID NO: 4 of WO 99/19467) and the 37 N-terminal amino acid residues ofthe alpha-amylase derived from Bacillus amyloliquefaciens (shown in SEQID NO: 5 of WO 99/19467). In a preferred embodiment this hybrid has oneor more, especially all, of the following substitutions:G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the Bacilluslicheniformis numbering in SEQ ID NO: 4 of WO 99/19467). Also preferredare variants having one or more of the following mutations (orcorresponding mutations in other Bacillus alpha-amylases): H154Y, A181T,N190F, A209V and Q264S and/or the deletion of two residues betweenpositions 176 and 179, preferably the deletion of E178 and G179 (usingSEQ ID NO: 5 of WO 99/19467 for position numbering).

In an embodiment the bacterial alpha-amylase is the mature part of thechimeric alpha-amylase disclosed in Richardson et al. (2002), TheJournal of Biological Chemistry, Vol. 277, No 29, Issue 19 July, pp.267501-26507, referred to as BD5088 or a variant thereof. Thisalpha-amylase is the same as the one shown in SEQ ID NO: 2 in WO2007134207. The mature enzyme sequence starts after the initial “Met”amino acid in position 1.

Thermostable Alpha-Amylase

According to the invention the alpha-amylase may be a thermostablealpha-amylase, such as a thermostable bacterial alpha-amylase,preferably from Bacillus stearothermophilus. In an embodiment thealpha-amylase used according to the invention has a T½ (min) at pH 4.5,85° C., 0.12 mM CaCl₂ of at least 10.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, of at least 15.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, of as at least 20.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, of as at least 25.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, of as at least 30.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, of as at least 40.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, of at least 50.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, of at least 60.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, between 10-70.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, between 15-70.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, between 20-70.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, between 25-70.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, between 30-70.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, between 40-70.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, between 50-70.

In an embodiment the thermostable alpha-amylase has a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂, between 60-70.

In an embodiment of the invention the alpha-amylase is an bacterialalpha-amylase, preferably derived from the genus Bacillus, especially astrain of Bacillus stearothermophilus, in particular the Bacillusstearothermophilus as disclosed in WO 99/019467 as SEQ ID NO: 3 (SEQ IDNO: 1 herein) with one or two amino acids deleted at positions R179,G180, I181 and/or G182, in particular with R179 and G180 deleted, orwith I181 and G182 deleted, with mutations in below list of mutations.

In preferred embodiments the Bacillus stearothermophilus alpha-amylaseshave double deletion I181+G182, and optional substitution N193F, furthercomprising mutations selected from below list:

-   -   V59A+Q89R+G112D+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+D269E+D281N;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+I270L;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+H274K;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+Y276F;    -   V59A+E129V+R157Y+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;    -   59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+H274K;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+D281N;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+G416V;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   A91L+M96I+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F+L427M;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+N376*+I377*;    -   E129V+K177L+R179E+K220P+N224L+Q254S;    -   E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   E129V+K177L+R179E+S242Q;    -   E129V+K177L+R179V+K220P+N224L+S242Q+Q254S;    -   K220P+N224L+S242Q+Q254S;    -   M284V;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V.    -   V59A+E129V+K177L+R179E+Q254S+M284V;

In a preferred embodiment the alpha-amylase is selected from the groupof Bacillus stearothermophilus alpha-amylase variants:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   I181*+G182*+N193F+V59A+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 herein for numbering).

It should be understood that when referring to Bacillusstearothermophilus alpha-amylase and variants thereof they are normallyproduced in truncated form. In particular, the truncation may be so thatthe Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 inWO 99/19467 or SEQ ID NO: 1 herein, or variants thereof, are truncatedin the C-terminal and are typically around 491 amino acids long, such asfrom 480-495 amino acids long.

In a preferred embodiment the alpha-amylase variant may be an enzymehaving a degree of identity of at least 60%, e.g., at least 70%, atleast 80%, at least 90%, at least 95%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99%, but less than 100% to the sequence shown inSEQ ID NO: 3 in WO 99/19467 or SEQ ID NO: 1 herein.

Protease Present and/or Added During Liquefaction

According to the invention a thermostable protease is optionally presentand/or added during liquefaction together with an alpha-amylase, andoptionally a carbohydrate-source generating enzyme, in particular athermostable glucoamylase, and/or optionally a pullulanase.

Proteases are classified on the basis of their catalytic mechanism intothe following groups: Serine proteases (S), Cysteine proteases (C),Aspartic proteases (A), Metallo proteases (M), and Unknown, or as yetunclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J.Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), inparticular the general introduction part.

In a preferred embodiment the thermostable protease used according tothe invention is a “metallo protease” defined as a protease belonging toEC 3.4.24 (metalloendopeptidases); preferably EC 3.4.24.39 (acid metalloproteinases).

To determine whether a given protease is a metallo protease or not,reference is made to the above “Handbook of Proteolytic Enzymes” and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

Protease activity can be measured using any suitable assay, in which asubstrate is employed, that includes peptide bonds relevant for thespecificity of the protease in question. Assay-pH and assay-temperatureare likewise to be adapted to the protease in question. Examples ofassay-pH-values are pH 6, 7, 8, 9, 10, or 11. Examples ofassay-temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80° C.

Examples of protease substrates are casein, such as Azurine-CrosslinkedCasein (AZCL-casein). Two protease assays are described below in the“Materials & Methods”-section, of which the so-called “AZCL-CaseinAssay” is the preferred assay.

In an embodiment the thermostable protease has at least 20%, such as atleast 30%, such as at least 40%, such as at least 50%, such as at least60%, such as at least 70%, such as at least 80%, such as at least 90%,such as at least 95%, such as at least 100% of the protease activity ofthe Protease 196 variant or Protease Pfu determined by the AZCL-caseinassay described in the “Materials & Methods” section.

There are no limitations on the origin of the protease used in a processof the invention as long as it fulfills the thermostability propertiesdefined below.

In one embodiment the protease is of fungal origin.

The protease may be a variant of, e.g., a wild-type protease as long asthe protease has the thermostability properties defined herein. In apreferred embodiment the thermostable protease is a variant of a metalloprotease as defined above. In an embodiment the thermostable proteaseused in a process of the invention is of fungal origin, such as a fungalmetallo protease, such as a fungal metallo protease derived from astrain of the genus Thermoascus, preferably a strain of Thermoascusaurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670(classified as EC 3.4.24.39).

In an embodiment the thermostable protease is a variant of the maturepart of the metallo protease shown in SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 andshown as SEQ ID NO: 3 herein further with mutations selected from belowlist:

-   -   S5*+D79L+S87P+A112P+D142L;    -   D79L+S87P+A112P+T124V+D142L;    -   S5*+N26R+D79L+S87P+A112P+D142L;    -   N26R+T46R+D79L+S87P+A112P+D142L;    -   T46R+D79L+S87P+T116V+D142L;    -   D79L+P81R+S87P+A112P+D142L;    -   A27K+D79L+S87P+A112P+T124V+D142L;    -   D79L+Y82F+S87P+A112P+T124V+D142L;    -   D79L+Y82F+S87P+A112P+T124V+D142L;    -   D79L+S87P+A112P+T124V+A126V+D142L;    -   D79L+S87P+A112P+D142L;    -   D79L+Y82F+S87P+A112P+D142L;    -   S38T+D79L+S87P+A112P+A126V+D142L;    -   D79L+Y82F+S87P+A112P+A126V+D142L;    -   A27K+D79L+S87P+A112P+A126V+D142L;    -   D79L+S87P+N98C+A112P+G135C+D142L;    -   D79L+S87P+A112P+D142L+T141C+M161C;    -   S36P+D79L+S87P+A112P+D142L;    -   A37P+D79L+S87P+A112P+D142L;    -   S49P+D79L+S87P+A112P+D142L;    -   S50P+D79L+S87P+A112P+D142L;    -   D79L+S87P+D104P+A112P+D142L;    -   D79L+Y82F+S87G+A112P+D142L;    -   S70V+D79L+Y82F+S87G+Y97W+A112P+D142L;    -   D79L+Y82F+S87G+Y97W+D104P+A112P+D142L;    -   S70V+D79L+Y82F+S87G+A112P+D142L;    -   D79L+Y82F+S87G+D104P+A112P+D142L;    -   D79L+Y82F+S87G+A112P+A126V+D142L;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L;    -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;    -   A27K+Y82F+S87G+D104P+A112P+A126V+D142L;    -   A27K+D79L+Y82F+D104P+A112P+A126V+D142L;    -   A27K+Y82F+D104P+A112P+A126V+D142L;    -   A27K+D79L+S87P+A112P+D142L;    -   D79L+S87P+D142L.

In an preferred embodiment the thermostable protease is a variant of themetallo protease disclosed as the mature part of SEQ ID NO: 2 disclosedin WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841or SEQ ID NO: 3 herein with the following mutations:

D79L+S87P+A112P+D142L; D79L+S87P+D142L; orA27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

In an embodiment the protease variant has at least 75% identitypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe mature part of the polypeptide of SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO: 1 in WO 2010/008841 or SEQID NO: 3 herein.

The thermostable protease may also be derived from any bacterium as longas the protease has the thermostability properties defined according tothe invention.

In an embodiment the thermostable protease is derived from a strain ofthe bacterium Pyrococcus, such as a strain of Pyrococcus furiosus (pfuprotease)

In an embodiment the protease is one shown as SEQ ID NO: 1 in U.S. Pat.No. 6,358,726-B1 (Takara Shuzo Company), SEQ ID NO: 13 herein or SEQ IDNO: 29 herein.

In another embodiment the thermostable protease is one disclosed in SEQID NO: 13 herein or a protease having at least 80% identity, such as atleast 85%, such as at least 90%, such as at least 95%, such as at least96%, such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 1 in U.S. Pat. No. 6,358,726-B1 or SEQ ID NO: 13herein. The Pyroccus furiosus protease can be purchased from Takara Bio,Japan.

The Pyrococcus furiosus protease is a thermostable protease according tothe invention. The commercial product Pyrococcus furiosus protease (PfuS) was found to have a thermostability of 110% (80° C./70° C.) and 103%(90° C./70° C.) at pH 4.5 determined as described in Example 2 herein.

In one embodiment a thermostable protease used in a process of theinvention has a thermostability value of more than 20% determined asRelative Activity at 80° C./70° C. determined as described in Example 2.

In an embodiment the protease has a thermostability of more than 30%,more than 40%, more than 50%, more than 60%, more than 70%, more than80%, more than 90%, more than 100%, such as more than 105%, such as morethan 110%, such as more than 115%, such as more than 120% determined asRelative Activity at 80° C./70° C.

In an embodiment protease has a thermostability of between 20 and 50%,such as between 20 and 40%, such as 20 and 30% determined as RelativeActivity at 80° C./70° C.

In an embodiment the protease has a thermostability between 50 and 115%,such as between 50 and 70%, such as between 50 and 60%, such as between100 and 120%, such as between 105 and 115% determined as RelativeActivity at 80° C./70° C.

In an embodiment the protease has a thermostability value of more than10% determined as Relative Activity at 85° C./70° C. determined asdescribed in Example 2.

In an embodiment the protease has a thermostability of more than 10%,such as more than 12%, more than 14%, more than 16%, more than 18%, morethan 20%, more than 30%, more than 40%, more that 50%, more than 60%,more than 70%, more than 80%, more than 90%, more than 100%, more than110% determined as Relative Activity at 85° C./70° C.

In an embodiment the protease has a thermostability of between 10 and50%, such as between 10 and 30%, such as between 10 and 25% determinedas Relative Activity at 85° C./70° C.

In an embodiment the protease has more than 20%, more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90% determined as Remaining Activity at 80° C.; and/or

In an embodiment the protease has more than 20%, more than 30%, morethan 40%, more than 50%, more than 60%, more than 70%, more than 80%,more than 90% determined as Remaining Activity at 84° C.

Determination of “Relative Activity” and “Remaining Activity” is done asdescribed in Example 2.

In an embodiment the protease may have a themostability for above 90,such as above 100 at 85° C. as determined using the Zein-BCA assay asdisclosed in Example 3.

In an embodiment the protease has a themostability above 60%, such asabove 90%, such as above 100%, such as above 110% at 85° C. asdetermined using the Zein-BCA assay.

In an embodiment protease has a themostability between 60-120, such asbetween 70-120%, such as between 80-120%, such as between 90-120%, suchas between 100-120%, such as 110-120% at 85° C. as determined using theZein-BCA assay.

In an embodiment the thermostable protease has at least 20%, such as atleast 30%, such as at least 40%, such as at least 50%, such as at least60%, such as at least 70%, such as at least 80%, such as at least 90%,such as at least 95%, such as at least 100% of the activity of theJTP196 protease variant or Protease Pfu determined by the AZCL-caseinassay.

Carbohydrate-Source Generating Enzyme Present and/or Added DuringLiquefaction

According to the invention a carbohydrate-source generating enzyme, inparticular a glucoamylase, preferably a thermostable glucoamylase, mayoptionally be present and/or added during liquefaction together with analpha-amylase and an optional thermostable protease. As mentioned above,a pullulanase may also be optionally be present and/or added duringliquefaction step i).

The term “carbohydrate-source generating enzyme” includes any enzymesgenerating fermentable sugars. A carbohydrate-source generating enzymeis capable of producing a carbohydrate that can be used as anenergy-source by the fermenting organism(s) in question, for instance,when used in a process of the invention for producing a fermentationproduct, such as ethanol. The generated carbohydrates may be converteddirectly or indirectly to the desired fermentation product, preferablyethanol. According to the invention a mixture of carbohydrate-sourcegenerating enzymes may be used. Specific examples include glucoamylase(being glucose generators), beta-amylase and maltogenic amylase (beingmaltose generators).

In a preferred embodiment the carbohydrate-source generating enzyme isthermostable. The carbohydrate-source generating enzyme, in particularthermostable glucoamylase, may be added together with or separately fromthe alpha-amylase and the thermostable protease.

In an embodiment the carbohydrate-source generating enzyme, preferably athermostable glucoamylase, has a Relative Activity heat stability at 85°C. of at least 20%, at least 30%, preferably at least 35% determined asdescribed in Example 4 (heat stability).

In an embodiment the carbohydrate-source generating enzyme is aglucoamylase having a relative activity pH optimum at pH 5.0 of at least90%, preferably at least 95%, preferably at least 97%, such as 100%determined as described in Example 4 (pH optimum).

In an embodiment the carbohydrate-source generating enzyme is aglucoamylase having a pH stability at pH 5.0 of at least at least 80%,at least 85%, at least 90% determined as described in Example 4 (pHstability).

In a specific and preferred embodiment the carbohydrate-sourcegenerating enzyme is a thermostable glucoamylase, preferably of fungalorigin, preferably a filamentous fungi, such as from a strain of thegenus Penicillium, especially a strain of Penicillium oxalicum, inparticular the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO:2 in PCT/CN10/071753 published as WO 2011/127802 (which is herebyincorporated by reference) and shown in SEQ ID NO: 9 or 14 herein.

In an embodiment the thermostable glucoamylase has at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99% or 100% identity to the mature polypeptide shown in SEQ ID NO:2 in WO 2011/127802 or SEQ ID NOs: 9 or 14 herein.

In a preferred embodiment the carbohydrate-source generating enzyme is avariant of the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO:2 in WO 2011/127802 and shown in SEQ ID NO: 9 and 14 herein, having aK79V substitution (using the mature sequence shown in SEQ ID NO: 14 fornumbering). The K79V glucoamylase variant has reduced sensitivity toprotease degradation relative to the parent as disclosed in co-pendingU.S. application No. 61/531,189 or PCT/US12/053779 (which are herebyincorporated by reference).

In an embodiment the carbohydrate-source generating enzyme, inparticular thermostable glucoamylase, is derived from Penicilliumoxalicum.

In an embodiment the thermostable glucoamylase is a variant of thePenicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO2011/127802 and shown in SEQ ID NO: 9 and 14 herein. In a preferredembodiment the Penicillium oxalicum glucoamylase is the one disclosed asSEQ ID NO: 2 in WO 2011/127802 and shown in SEQ ID NO: 9 and 14 hereinhaving Val (V) in position 79 (using SEQ ID NO: 14 for numbering).

Contemplated Penicillium oxalicum glucoamylase variants are disclosed inco-pending PCT application # PCT/EP12/070127 (which is herebyincorporated by reference).

In an embodiment these variants have reduced sensitivity to proteasedegradation.

In an embodiment these variant have improved thermostability compared tothe parent.

More specifically, in an embodiment the glucoamylase has a K79Vsubstitution (using SEQ ID NO: 14 for numbering), corresponding to thePE001 variant, and further comprises at least one of the followingsubstitutions or combination of substitutions:

T65A; or Q327F; or E501V; or Y504T; or Y504*; or T65A+Q327F; orT65A+E501V; or T65A+Y504T; or T65A+Y504*; or Q327F+E501V; orQ327F+Y504T; or Q327F+Y504*; or E501V+Y504T; or E501V+Y504*; orT65A+Q327F+E501V; or T65A+Q327F+Y504T; or T65A+E501V+Y504T; orQ327F+E501V+Y504T; or T65A+Q327F+Y504*; or T65A+E501V+Y504*; orQ327F+E501V+Y504*; or T65A+Q327F+E501V+Y504T; or T65A+Q327F+E501V+Y504*;E501V+Y504T; or T65A+K161S; or T65A+Q405T; or T65A+Q327W; or T65A+Q327F;or T65A+Q327Y; or P11F+T65A+Q327F; orR1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F; or P2N+P4S+P11F+T65A+Q327F; orP11F+D26C+K33C+T65A+Q327F; or P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; orR1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; or P11F+T65A+Q327W; orP2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or P11F+T65A+Q327W+E501V+Y504T; orT65A+Q327F+E501V+Y504T; or T65A+S105P+Q327W; or T65A+S105P+Q327F; orT65A+Q327W+S364P; or T65A+Q327F+S364P; or T65A+S103N+Q327F; orP2N+P4S+P11F+K34Y+T65A+Q327F; or P2N+P4S+P11F+T65A+Q327F+D445N+V447S; orP2N+P4S+P11F+T65A+I172V+Q327F; or P2N+P4S+P11F+T65A+Q327F+N502*; orP2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E; orP2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S; orP2N+P4S+P11F+T65A+Q327F+S377T; or P2N+P4S+P11F+T65A+V325T+Q327W; orP2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T; orP2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T; orP2N+P4S+P11F+D26N+K34Y+T65A+Q327F; orP2N+P4S+P11F+T65A+Q327F+1375A+E501V+Y504T; orP2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; orP2N+P4S+T10D+T65A+Q327F+E501V+Y504T; orP2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; or K5A+P11F+T65A+Q327F+E501V+Y504T;or P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T; orP2N+T10E+E18N+T65A+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N; orP2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A; orP2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T; orP2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T; orP2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; orP2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*; orP2N+P4S+P11F+T65A+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; orK5A+P11F+T65A+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; orP2N+P4S+P11F+T65A+V79A+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+V79G+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+V791+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+V79L+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+V79S+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; or S255N+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T.

In a preferred embodiment the Penicillium oxalicum glucoamylase varianthas a K79V substitution (using SEQ ID NO: 14 for numbering),corresponding to the PE001 variant, and further comprises one of thefollowing mutations:

P11F+T65A+Q327F; or P2N+P4S+P11F+T65A+Q327F; orP11F+D26C+K33C+T65A+Q327F; or P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; orP2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or P11F+T65A+Q327W+E501V+Y504T.

The carbohydrate-source generating enzyme, in particular, may be addedin amounts from 0.1-100 micrograms EP/g, such as 0.5-50 micrograms EP/g,such as 1-25 micrograms EP/g, such as 2-12 micrograms EP/g DS.Pullulanase Present and/or Added During

Liquefaction

Optionally a pullulanase may be present and/or added during liquefactionstep i) together with an alpha-amylase and optionally a thermostableprotease and/or carbohydrate-source generating enzyme. As mentionedabove a carbohydrate-source generating enzyme, preferably a thermostableglucoamylase, may also be present and/or added during liquefaction stepi).

The pullulanase may be present and/or added during liquefaction step i)and/or saccharification step ii) or simultaneous saccharification andfermentation.

Pullulanases (E.C. 3.2.1.41, pullulan 6-glucano-hydrolase), aredebranching enzymes characterized by their ability to hydrolyze thealpha-1,6-glycosidic bonds in, for example, amylopectin and pullulan.

Contemplated pullulanases according to the present invention include thepullulanases from Bacillus amyloderamificans disclosed in U.S. Pat. No.4,560,651 (hereby incorporated by reference), the pullulanase disclosedas SEQ ID NO: 2 in WO 01/151620 (hereby incorporated by reference), theBacillus deramificans disclosed as SEQ ID NO: 4 in WO 01/151620 (herebyincorporated by reference), and the pullulanase from Bacillusacidopullulyticus disclosed as SEQ ID NO: 6 in WO 01/151620 (herebyincorporated by reference) and also described in FEMS Mic. Let. (1994)115, 97-106.

Additional pullulanases contemplated according to the present inventionincluded the pullulanases from Pyrococcus woesei, specifically fromPyrococcus woesei DSM No. 3773 disclosed in WO92/02614.

In an embodiment the pullulanase is a family GH57 pullulanase. In anembodiment the pullulanase includes an X47 domain as disclosed in U.S.61/289,040 published as WO 2011/087836 (which are hereby incorporated byreference). More specifically the pullulanase may be derived from astrain of the genus Thermococcus, including Thermococcus litoralis andThermococcus hydrothermalis, such as the Thermococcus hydrothermalispullulanase shown in SEQ ID NO: 11 truncated at site X4 right after theX47 domain (i.e., amino acids 1-782 in SEQ ID NOS: 11 and 12 herein).The pullulanase may also be a hybrid of the Thermococcus litoralis andThermococcus hydrothermalis pullulanases or a T. hydrothermalis/T.litoralis hybrid enzyme with truncation site X4 disclosed in U.S.61/289,040 published as WO 2011/087836 (which is hereby incorporated byreference) and disclosed in SEQ ID NO: 12 herein.

In another embodiment the pullulanase is one comprising an X46 domaindisclosed in WO 2011/076123 (Novozymes).

The pullulanase may according to the invention be added in an effectiveamount which include the preferred amount of about 0.0001-10 mg enzymeprotein per gram DS, preferably 0.0001-0.10 mg enzyme protein per gramDS, more preferably 0.0001-0.010 mg enzyme protein per gram DS.Pullulanase activity may be determined as NPUN. An Assay fordetermination of NPUN is described in the “Materials & Methods”-sectionbelow.

Suitable commercially available pullulanase products include PROMOZYMED, PROMOZYME™ D2 (Novozymes NS, Denmark), OPTIMAX L-300(DuPont-Genencor, USA), and AMANO 8 (Amano, Japan).

Carbohydrate-Source Generating Enzyme Present and/or Added DuringSaccharification and/or Fermentation

According to the invention a carbohydrate-source generating enzyme,preferably a glucoamylase, may be present and/or added duringsaccharification and/or fermentation.

In a preferred embodiment the carbohydrate-source generating enzyme is aglucoamylase, of fungal origin, preferably from a stain of Aspergillus,preferably A. niger, A. awamori, or A. oryzae; or a strain ofTrichoderma, preferably T. reesei; or a strain of Talaromyces,preferably T. emersonii,

Glucoamylase

According to the invention the glucoamylase present and/or added duringsaccharification and/or fermentation may be derived from any suitablesource, e.g., derived from a microorganism or a plant. Preferredglucoamylases are of fungal or bacterial origin, selected from the groupconsisting of Aspergillus glucoamylases, in particular Aspergillus nigerG1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102),or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylasedisclosed in WO 84/02921, Aspergillus oryzae glucoamylase (Agric. Biol.Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof.Other Aspergillus glucoamylase variants include variants with enhancedthermal stability: G137A and G139A (Chen et al. (1996), Prot. Eng. 9,499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Eng. 8, 575-582);N182 (Chen et al. (1994), Biochem. J. 301, 275-281); disulphide bonds,A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; andintroduction of Pro residues in position A435 and S436 (Li et al.(1997), Protein Eng. 10, 1199-1204.

Other glucoamylases include Athelia rolfsii (previously denotedCorticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and(Nagasaka et al. (1998) “Purification and properties of theraw-starch-degrading glucoamylases from Corticium rolfsii, ApplMicrobiol Biotechnol 50:323-330), Talaromyces glucoamylases, inparticular derived from Talaromyces emersonii (WO 99/28448), Talaromycesleycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, Talaromycesthermophilus (U.S. Pat. No. 4,587,215). In a preferred embodiment theglucoamylase used during saccharification and/or fermentation is theTalaromyces emersonii glucoamylase disclosed in WO 99/28448.

Bacterial glucoamylases contemplated include glucoamylases from thegenus Clostridium, in particular C. thermoamylolyticum (EP 135,138), andC. thermohydrosulfuricum (WO 86/01831).

Contemplated fungal glucoamylases include Trametes cingulata,Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed inWO 2006/069289; or Peniophora rufomarginata disclosed in WO2007/124285;or a mixture thereof. Also hybrid glucoamylase are contemplatedaccording to the invention. Examples include the hybrid glucoamylasesdisclosed in WO 2005/045018. Specific examples include the hybridglucoamylase disclosed in Table 1 and 4 of Example 1 (which hybrids arehereby incorporated by reference).

In an embodiment the glucoamylase is derived from a strain of the genusPycnoporus, in particular a strain of Pycnoporus as described in WO2011/066576 (SEQ ID NOs 2, 4 or 6), such as SEQ ID NO: 28 herein, orfrom a strain of the genus Gloeophyllum, such as a strain ofGloeophyllum sepiarium or Gloeophyllum trabeum, in particular a strainof Gloeophyllum as described in WO 2011/068803 (SEQ ID NO: 2, 4, 6, 8,10, 12, 14 or 16). In a preferred embodiment the glucoamylase is SEQ IDNO: 2 in WO 2011/068803 or SEQ ID NO: 26 herein.

In a preferred embodiment the glucoamylase is SEQ ID NO: 27 herein. Inan embodiment the glucoamylase is derived from a strain of the genusNigrofomes, in particular a strain of Nigrofomes sp. disclosed in WO2012/064351 (SEQ ID NO: 2) (all references hereby incorporated byreference). Contemplated are also glucoamylases which exhibit a highidentity to any of the above mentioned glucoamylases, i.e., at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100%identity to any one of the mature parts of the enzyme sequencesmentioned above, such as any of SEQ ID NOs: 26, 27, 28 or 29 herein,preferably SEQ ID NO: 26 herein.

Glucoamylases may in an embodiment be added to the saccharificationand/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2AGU/g DS.

In an embodiment the glucoamylase is added as a blend further comprisingan alpha-amylase. In a preferred embodiment the alpha-amylase is afungal alpha-amylase, especially an acid fungal alpha-amylase. Thealpha-amylase is typically a side activity.

In an embodiment the glucoamylase is a blend comprising Talaromycesemersonii glucoamylase disclosed in WO 99/28448 as SEQ ID NO: 7 andTrametes cingulata glucoamylase disclosed in WO 06/069289.

In an embodiment the glucoamylase is a blend comprising Talaromycesemersonii glucoamylase disclosed in WO 99/28448, Trametes cingulataglucoamylase disclosed in WO 06/69289, and Rhizomucor pusillusalpha-amylase with Aspergillus niger glucoamylase linker and SBDdisclosed as V039 in Table 5 in WO 2006/069290.

In an embodiment the glucoamylase is a blend comprising Talaromycesemersonii glucoamylase disclosed in WO99/28448, Trametes cingulataglucoamylase disclosed in WO 06/69289, and Rhizomucor pusillusalpha-amylase with Aspergillus niger glucoamylase linker and SBDdisclosed as V039 in Table 5 in WO 2006/069290.

In an embodiment the glucoamylase is a blend comprising Gloeophyllumsepiarium glucoamylase shown as SEQ ID NO: 2 in WO 2011/068803 andRhizomucor pusillus with an Aspergillus niger glucoamylase linker andstarch-binding domain (SBD), disclosed SEQ ID NO: 3 in WO 2013/006756with the following substitutions: G128D+D143N.

In an embodiment the alpha-amylase may be derived from a strain of thegenus Rhizomucor, preferably a strain the Rhizomucor pusillus, such asthe one shown in SEQ ID NO: 3 in WO2013/006756, or the genus Meripilus,preferably a strain of Meripilus giganteus.

In a preferred embodiment the alpha-amylase is derived from a Rhizomucorpusillus with an Aspergillus niger glucoamylase linker andstarch-binding domain (SBD), disclosed as V039 in Table 5 in WO2006/069290.

In an embodiment the Rhizomucor pusillus alpha-amylase or the Rhizomucorpusillus alpha-amylase with an Aspergillus niger glucoamylase linker andstarch-binding domain (SBD) has at least one of the followingsubstitutions or combinations of substitutions: D165M; Y141W; Y141R;K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W;G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R;Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N;Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C;Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C;G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C (using SEQ IDNO: 3 in WO 2013/006756 for numbering). In a preferred embodiment theglucoamylase blend comprises Gloeophyllum sepiarium glucoamylase (e.g.,SEQ ID NO: 2 in WO 2011/068803) and Rhizomucor pusillus alpha-amylase.

In a preferred embodiment the glucoamylase blend comprises Gloeophyllumsepiarium glucoamylase shown as SEQ ID NO: 2 in WO 2011/068803 andRhizomucor pusillus with an Aspergillus niger glucoamylase linker andstarch-binding domain (SBD), disclosed SEQ ID NO: 3 in WO 2013/006756with the following substitutions: G128D+D143N

Commercially available compositions comprising glucoamylase include AMG200 L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL, SPIRIZYMEACHIEVE and AMG™ E (from Novozymes NS); OPTIDEX™ 300, GC480, GC417 (fromDuPont-Genencor); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900,G-ZYME™ and G990 ZR (from DuPont-Genencor).

Maltogenic Amylase

The carbohydrate-source generating enzyme present and/or added duringsaccharification and/or fermentation may also be a maltogenicalpha-amylase. A “maltogenic alpha-amylase” (glucan1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amyloseand amylopectin to maltose in the alpha-configuration. A maltogenicamylase from Bacillus stearothermophilus strain NCIB 11837 iscommercially available from Novozymes NS. Maltogenic alpha-amylases aredescribed in U.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, whichare hereby incorporated by reference. The maltogenic amylase may in apreferred embodiment be added in an amount of 0.05-5 mg totalprotein/gram DS or 0.05-5 MANU/g DS.

Cellulolytic Composition Present and/or Added During Saccharificationand/or Fermentation

According to the invention a cellulolytic composition is present duringfermentation or simultaneous saccharification and fermentation (SSF).

The cellulolytic composition may be any cellulolytic composition,comprising a beta-glucosidase, a cellobiohydrolase and an endoglucanase.

Examples of suitable cellulolytic composition can be found in WO2008/151079 and co-pending patent application PCT/US12/052163 publishedas WO 2013/028928 which are incorporated by reference.

In preferred embodiments the cellulolytic composition is derived from astrain of Trichoderma, Humicola, or Chrysosporium.

In an embodiment the cellulolytic composition is derived from a strainof Trichoderma reesei, Humicola insolens and/or Chrysosporiumlucknowense.

In an embodiment the cellulolytic composition comprises abeta-glucosidase, preferably one derived from a strain of the genusAspergillus, such as Aspergillus oryzae, such as the one disclosed in WO2002/095014 or the fusion protein having beta-glucosidase activitydisclosed in WO 2008/057637, or Aspergillus fumigatus, such as onedisclosed in WO 2005/047499 or SEQ ID NO: 22 herein or an Aspergillusfumigatus beta-glucosidase variant disclosed in WO 2012/044915(Novozymes), such as one with the following substitutions F100D, S283G,N456E, F512Y; or a strain of the genus a strain Penicillium, such as astrain of the Penicillium brasilianum disclosed in WO 2007/019442, or astrain of the genus Trichoderma, such as a strain of Trichoderma reesei.

In an embodiment the cellulolytic composition comprises a GH61polypeptide having cellulolytic enhancing activity such as one derivedfrom the genus Thermoascus, such as a strain of Thermoascus aurantiacus,such as the one described in WO 2005/074656 as SEQ ID NO: 2; or onederived from the genus Thielavia, such as a strain of Thielaviaterrestris, such as the one described in WO 2005/074647 as SEQ ID NO: 7and SEQ ID NO: 8; or one derived from a strain of Aspergillus, such as astrain of Aspergillus fumigatus, such as the one described in WO2010/138754 as SEQ ID NO: 1 and SEQ ID NO: 2; or one derived from astrain derived from Penicillium, such as a strain of Penicilliumemersonii, such as the one disclosed in WO 2011/041397 or SEQ ID NO: 23herein.

In an embodiment the cellulolytic composition comprises acellobiohydrolase I (CBH I), such as one derived from a strain of thegenus Aspergillus, such as a strain of Aspergillus fumigatus, such asthe Cel7a CBHI disclosed in SEQ ID NO: 2 in WO 2011/057140 or SEQ ID NO:24 herein, or a strain of the genus Trichoderma, such as a strain ofTrichoderma reesei.

In an embodiment the cellulolytic composition comprises acellobiohydrolase II (CBH II, such as one derived from a strain of thegenus Aspergillus, such as a strain of Aspergillus fumigatus or SEQ IDNO: 25 herein; or a strain of the genus Trichoderma, such as Trichodermareesei, or a strain of the genus Thielavia, such as a strain ofThielavia terrestris, such as cellobiohydrolase II CEL6A from Thielaviaterrestris.

In an embodiment the cellulolytic composition comprises a GH61polypeptide having cellulolytic enhancing activity and abeta-glucosidase.

In an embodiment the cellulolytic composition comprises a GH61polypeptide having cellulolytic enhancing activity, a beta-glucosidase,and a CBH I.

In an embodiment the cellulolytic composition comprises a GH61polypeptide having cellulolytic enhancing activity, a beta-glucosidase,a CBH I, and a CBH II.

In an embodiment the cellulolytic composition is a Trichoderma reeseicellulolytic enzyme composition, further comprising Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity(SEQ ID NO: 2 in WO 2005/074656), and Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637).

In an embodiment the cellulolytic composition is a Trichoderma reeseicellulolytic enzyme composition, further comprising Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity(SEQ ID NO: 2 in WO 2005/074656) and Aspergillus fumigatusbeta-glucosidase (SEQ ID NO: 2 of WO 2005/047499) or SEQ ID NO: 22herein.

In an embodiment the cellulolytic composition is a Trichoderma reeseicellulolytic enzyme composition further comprising Penicillium emersoniiGH61A polypeptide having cellulolytic enhancing activity disclosed in WO2011/041397 and Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 ofWO 2005/047499) or SEQ ID NO: 22 herein or a variant thereof with thefollowing substitutions F100D, S283G, N456E, F512Y.

In a preferred embodiment the cellulolytic composition comprising one ormore of the following components:

(i) an Aspergillus fumigatus cellobiohydrolase I;

(ii) an Aspergillus fumigatus cellobiohydrolase II;

(iii) an Aspergillus fumigatus beta-glucosidase or variant thereof; and

(iv) a Penicillium sp. GH61 polypeptide having cellulolytic enhancingactivity; or homologs thereof.

In an preferred embodiment the cellulolytic composition is derived fromTrichoderma reesei comprising GH61A polypeptide having cellulolyticenhancing activity derived from a strain of Penicillium emersonii (SEQID NO: 2 in WO 2011/041397 or SEQ ID NO: 23 herein), Aspergillusfumigatus beta-glucosidase (SEQ ID NO: 2 in WO 2005/047499 SEQ ID NO: 22herein) variant F100D, S283G, N456E, F512Y) disclosed in WO 2012/044915;Aspergillus fumigatus Cel7A CBH1 disclosed as SEQ ID NO: 6 inWO2011/057140 (SEQ ID NO: 24 herein) and Aspergillus fumigatus CBH IIdisclosed as SEQ ID NO: 18 in WO 2011/057140 (SEQ ID NO: 25 herein).

In an embodiment the cellulolytic composition is dosed from 0.0001-3 mgEP/g DS, preferably, 0.0005-2 mg EP/g DS, preferably 0.001-1 mg/g DS,more preferably 0.005-0.5 mg EP/g DS, and even more preferably 0.01-0.1mg EP/g DS.

Examples of Preferred Processes of the Invention

In a preferred embodiment the process of the invention relates to aprocess for producing fermentation products from starch-containingmaterial comprising the steps of:

i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.,        preferably derived from Pyrococcus furiosus and/or Thermoascus        aurantiacus; and    -   optionally a Penicillium oxalicum glucoamylase;

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.

In another preferred embodiment the process of the invention relates toa process for producing fermentation products from starch-containingmaterial comprising the steps of:

i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   optionally a protease, preferably derived from Pyrococcus        furiosus and/or

Thermoascus aurantiacus, having a thermostability value of more than 20%determined as Relative Activity at 80° C./70° C.;

-   -   optionally a Penicillium oxalicum glucoamylase;

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.

In another preferred embodiment the process of the invention relates toa process for producing fermentation products from starch-containingmaterial comprising the steps of:

i) liquefying the starch-containing material at a temperature between80-90° C.:

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   optionally a protease, preferably derived from Pyrococcus        furiosus and/or Thermoascus aurantiacus, having a        thermostability value of more than 20% determined as Relative        Activity at 80° C./70° C.; and    -   optionally a Penicillium oxalicum glucoamylase

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.

In another preferred embodiment the process of the invention relates toa process for producing fermentation products from starch-containingmaterial comprising the steps of:

i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182 and optional substitution N193F; and        optionally further one of the following set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   V59A+E129V+K177L+R179E+Q254S+M284V;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering).    -   optionally a protease, preferably derived from Pyrococcus        furiosus and/or Thermoascus aurantiacus, having a        thermostability value of more than 20% determined as Relative        Activity at 80° C./70° C.; and    -   optionally a Penicillium oxalicum glucoamylase shown in SEQ ID        NO: 14 having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering);

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.

In another preferred embodiment the process of the invention relates toa process for producing fermentation products from starch-containingmaterial comprising the steps of:

i) liquefying the starch-containing material at a temperature between80-90° C. using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182 and optional substitution N193F; and        optionally further one of the following set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   V59A+E129V+K177L+R179E+Q254S+M284V;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering).    -   optionally a protease, preferably derived from Pyrococcus        furiosus and/or Thermoascus aurantiacus having a thermostability        value of more than 20% determined as Relative Activity at 80°        C./70° C.;    -   optionally a pullulanase    -   optionally a Penicillium oxalicum glucoamylase shown in SEQ ID        NO: 14 having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering);

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.

In another preferred embodiment the process of the invention relates toa process for producing fermentation products from starch-containingmaterial comprising the steps of:

i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus;    -   optionally a protease, preferably derived from Pyrococcus        furiosus or Thermoascus aurantiacus, having a thermostability        value of more than 20% determined as Relative Activity at 80°        C./70° C.; and    -   optionally a pullulanase;    -   optionally a Penicillium oxalicum glucoamylase;

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.

In another preferred embodiment the process of the invention relates toa process for producing fermentation products from starch-containingmaterial comprising the steps of:

i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   optionally a protease, preferably derived from Pyrococcus        furiosus or Thermoascus aurantiacus, having a thermostability        value of more than 20% determined as Relative Activity at 80°        C./70° C.;    -   optionally a pullulanase;    -   optionally a Penicillium oxalicum glucoamylase;

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.

In another preferred embodiment the process of the invention relates toa process for producing fermentation products from starch-containingmaterial comprising the steps of:

i) liquefying the starch-containing material at a temperature between80-90° C.:

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl2 of at least 10;    -   optionally a protease, preferably derived from Pyrococcus        furiosus or Thermoascus aurantiacus, having a thermostability        value of more than 20% determined as Relative Activity at 80°        C./70° C.;    -   optionally a pullulanase;    -   optionally a Penicillium oxalicum glucoamylase;

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.

In another preferred embodiment the process of the invention relates toa process for producing fermentation products from starch-containingmaterial comprising the steps of:

i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182 and optional substitution N193F; and        optionally further one of the following set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   V59A+E129V+K177L+R179E+Q254S+M284V;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering);    -   optionally a protease, derived from Pyrococcus furiosus and/or        Thermoascus aurantiacus, having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.; and    -   optionally a pullulanase;    -   optionally a Penicillium oxalicum glucoamylase shown in SEQ ID        NO: 14 having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering);

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.

In another preferred embodiment the process of the invention relates toa process for producing fermentation products from starch-containingmaterial comprising the steps of:

i) liquefying the starch-containing material at a temperature between80-90° C. using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182 and optional substitution N193F; and        optionally further one of the following set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   V59A+E129V+K177L+R179E+Q254S+M284V;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering).    -   optionally a protease, derived from Pyrococcus furiosus and/or        Thermoascus aurantiacus, having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.; and        optionally    -   optionally a pullulanase;    -   optionally a Penicillium oxalicum glucoamylase shown in SEQ ID        NO: 14 having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering);

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.

In an embodiment the process of the invention comprises the steps of:

-   -   i) liquefying the starch-containing material at a temperature        between 80-90° C. at a pH between 5.0 and 6.5 using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion I181+G182+N193F; and optionally            further one of the following set of substitutions:        -   E129V+K177L+R179E;        -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;        -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;        -   V59A+E129V+K177L+R179E+Q254S+M284V        -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO:            1 herein for numbering).        -   a protease derived from Pyrococcus furiosus, preferably the            one shown in SEQ ID NO: 13 herein or 29 herein;        -   a Penicillium oxalicum glucoamylase shown in SEQ ID NO: 14            having substitutions selected from the group of:        -   K79V;        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F; or        -   K79V+P11F+D26C+K33C+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or        -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering);    -   ii) saccharifying using a glucoamylase enzyme;    -   iii) fermenting using a fermenting organism;        wherein a cellulolytic composition, such as a Trichoderma reesei        cellulolytic composition, is present and/or added during        fermentation or simultaneous saccharification and fermentation,        in particular a Trichoderma reesei cellulolytic composition        comprising one or more polypeptides selected from the group        consisting of:    -   GH61 polypeptide having cellulolytic enhancing activity,    -   beta-glucosidase;    -   Cellobiohydrolase I;    -   Cellobiohydrolase II;        or a mixture of two, three, or four thereof.

In an embodiment the invention relates to processes, comprising thesteps of:

-   -   i) liquefying the starch-containing material at a temperature        between 80-90° C. at a pH between 5.0 and 6.5 using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion            I181+G182+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V            (using SEQ ID NO: 1 herein for numbering).        -   a protease derived from Pyrococcus furiosus, preferably the            one shown in SEQ ID NO: 13 herein or SEQ ID NO: 29 herein;        -   a Penicillium oxalicum glucoamylase shown in SEQ ID NO: 14            having substitutions selected from the group of:        -   K79V+P11F+T65A+Q327F        -   K79V+P2N+P4F+P11F+T65A+Q327F (using SEQ ID NO: 14 for            numbering).    -   ii) saccharifying using a glucoamylase enzyme selected from the        group of Talaromyces emersonii glucoamylase or Gloeophyllum        serpiarium glucoamylase;    -   iii) fermenting using a Saccharomyces cerevisiae yeast        wherein a Trichoderma reesei cellulolytic composition is present        and/or added during fermentation or simultaneous        saccharification and fermentation.

In an embodiment the pullulanase present and/or added duringliquefaction step i) is a family GH57 pullulanase, wherein thepullulanase preferably includes an X47 domain as disclosed in WO2011/087836.

In another embodiment the pullulanase is derived from a strain from thegenus Thermococcus, including Thermococcus litoralis and Thermococcushydrothermalis, or a hybrid thereof.

In an embodiment the pullulanase is truncated Thermococcushydrothermalis pullulanase at site X4 or a T. hydrothermalis/T.litoralis hybrid enzyme with truncation site X4 disclosed in WO2011/087836 or shown in SEQ ID NO: 12 herein.

In an embodiment the Bacillus stearothermophilus alpha-amylase (SEQ IDNO: 1 herein) is the mature alpha-amylase or corresponding maturealpha-amylases having at least 80% identity, at least 90% identity, atleast 95% identity at least 96% identity at least 97% identity at least99% identity to the SEQ ID NO: 1.

In an embodiment the Pyrococcus furiosus protease (SEQ ID NO: 13 hereinor SEQ ID NO: 29 herein) and/or Thermoascus aurantiacus protease (SEQ IDNO: 3) are the mature proteases or corresponding mature proteases havingat least 80% identity, at least 90% identity, at least 95% identity atleast 96% identity at least 97% identity at least 99% identity to theSEQ ID NO: 13 or SEQ ID NO: 29 herein, or SEQ ID NO: 3, respectively.

In an embodiment the Penicillium oxalicum glucoamylase (SEQ ID NO: 14herein) is the mature glucoamylase or corresponding mature glucoamylasehaving at least 80% identity, at least 90% identity, at least 95%identity at least 96% identity at least 97% identity at least 99%identity to the SEQ ID NO: 14 herein.

A Composition Comprising Alpha-Amylase and Protease

A composition of the invention comprises an alpha-amylase and athermostable protease. The composition may also further comprise athermostable carbohydrate-source generating enzyme and/or optionally apullulanase too.

Therefore, in this aspect the invention relates to compositioncomprising:

i) an alpha-amylase;

ii) a protease has a thermostability value of more than 20% determinedas Relative Activity at 80° C./70° C.; and optionally

iii) a carbohydrate-source generating enzyme.

Alpha-Amylase:

The alpha-amylase may be any alpha-amylase, such as bacterialalpha-amylases, such as alpha-amylases derived from the genus Bacillus,such as Bacillus stearothermophilus.

The alpha-amylase may be a thermostable alpha-amylase. The thermostablealpha-amylase may have a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) ofat least 10, such as at least 15, such as at least 20, such as at least25, such as at least 30, such as at least 40, such as at least 50, suchas at least 60, such as between 10-70, such as between 15-70, such asbetween 20-70, such as between 25-70, such as between 30-70, such asbetween 40-70, such as between 50-70, such as between 60-70.

In an embodiment the alpha-amylase is selected from the group ofBacillus stearothermophilus alpha-amylase variants, in particulartruncated to be 491 amino acids long, such as from 480 to 495 aminoacids long, with mutations selected from the group of:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   I181*+G182*+N193F+V59A+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 herein for numbering).

It should be understood that these alpha-amylases are only specificexamples. Any alpha-amylase disclosed above in the “Alpha-AmylasePresent and/or Added During Liquefaction”-section above may be used asthe alpha-amylase component in a composition of the invention.

Protease:

A composition of the invention comprises a thermostable protease.

There is no limitation on the origin of the protease component as longas it fulfills the thermostability properties defined herein.

In a preferred embodiment the protease is a variant of the Thermoascusaurantiacus protease mentioned above having a thermostability value ofmore than 20% determined as Relative Activity at 80° C./70° C.determined as described in Example 2.

In a specific preferred embodiment the protease is a variant of themetallo protease derived from Thermoascus aurantiacus disclosed as themature part of SEQ ID NO. 2 disclosed in WO 2003/048353 or the maturepart of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 3 herein withmutations selected from the group of:

-   -   D79L+S87P+A112P+D142L;    -   D79L+S87P+D142L; and    -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

In another preferred embodiment the protease is derived from a strain ofPyrococcus furiosus, such as the one shown in SEQ ID NO: 1 in U.S. Pat.No. 6,358,726, SEQ ID NO: 13 herein or SEQ ID NO: 29 herein.

It should be understood that these proteases are only examples. Anyprotease disclosed above in the “Protease Present and/or Added DuringLiquefaction” section above may be used as the protease component in acomposition of the invention.

Carbohydrate-Source Generating Enzymes:

A composition of the invention may further comprise acarbohydrate-source generating enzyme, in particular a glucoamylase,which has a heat stability at 85° C., pH 5.3, of at least 30%,preferably at least 35%.

Said carbohydrate-source generating enzyme may be a thermostableglucoamylase having a Relative Activity heat stability at 85° C. of atleast 20%, at least 30%, preferably at least 35% determined as describedin Example 4 (Heat stability).

In an embodiment the carbohydrate-source generating enzyme is aglucoamylase having a relative activity pH optimum at pH 5.0 of at least90%, preferably at least 95%, preferably at least 97%, such as 100%determined as described in Example 4 (pH optimum).

In an embodiment the carbohydrate-source generating enzyme is aglucoamylase having a pH stability at pH 5.0 of at least at least 80%,at least 85%, at least 90% determined as described in Example 4 (pHstability).

In a preferred embodiment the carbohydrate-source generating enzyme is athermostable glucoamylase, preferably of fungal origin, preferably afilamentous fungi, such as from a strain of the genus Penicillium,especially a strain of Penicillium oxalicum disclosed as SEQ ID NO: 2 inPCT/CN10/071753 published as WO 2011/127802 (which is herebyincorporated by reference), or a variant thereof, and shown in SEQ IDNO: 9 or 14 herein.

In an embodiment the glucoamylase, or a variant thereof, may have atleast 80%, more preferably at least 85%, more preferably at least 90%,more preferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99% or 100% identity to the mature polypeptide shownin SEQ ID NO: 2 in WO 2011/127802 or SEQ ID NO: 9 or 14 herein.

In a specific and preferred embodiment the carbohydrate-sourcegenerating enzyme is a variant of the Penicillium oxalicum glucoamylasedisclosed as SEQ ID NO: 2 in WO 2011/127802 and shown in SEQ ID NO: 9and 14 herein, having a K79V substitution (using the mature sequenceshown in SEQ ID NO: 14 for numbering). The K79V glucoamylase variant hasreduced sensitivity to protease degradation relative to the parent asdisclosed in co-pending U.S. application No. 61/531,189 published as WO2013/036526 (which is hereby incorporated by reference).

Examples of suitable thermostable Penicillium oxalicum glucoamylasevariants are listed above and in Examples 15 and 16 below or Examples 10and 11 in WO 2013/053801 (hereby incorporated by reference).

In an embodiment the carbohydrate-source generating enzyme haspullulanase side activity.

It should be understood that these carbohydrate-source generatingenzymes, in particular glucoamylases, are only examples. Anycarbohydrate-source generating enzyme disclosed above in the“Carbohydrate-source generating enzyme Present and/or Added DuringLiquefaction” section above may be used as component in a composition ofthe invention.

Pullulanase:

A composition of the invention may further comprise a pullulanase. In anembodiment the pullulanase is a family GH57 pullulanase In a preferredembodiment the pullulanase includes an X47 domain as disclosed in U.S.61/289,040 published as WO 2011/087836 (which are hereby incorporated byreference).

Specifically the pullulanase may be derived from a strain from the genusThermococcus, including Thermococcus litoralis and Thermococcushydrothermalis or a hybrid thereof.

The pullulanase may be Thermococcus hydrothermalis pullulanase truncatedat site X4 or a Thermococcus hydrothermalis/T. litoralis hybrid enzymewith truncation site X4 as disclosed in U.S. 61/289,040 published as WO2011/087836.

In another embodiment the pullulanase is one comprising an X46 domaindisclosed in WO 2011/076123 (Novozymes).

It should be understood that these pullulanases are only specificexamples. Any pullulanase disclosed above in the “Pullulanase Presentand/or Added During Liquefaction” section above may be used as theoptional pullulanase component in a composition of the invention.

Preferred Compositions of the Invention

In a preferred embodiment the composition of the invention comprising

-   -   an alpha-amylase derived from Bacillus stearothermophilus;    -   optionally a protease, preferably derived from Pyrococcus        furiosus and/or Thermoascus aurantiacus, having a        thermostability value of more than 20% determined as Relative        Activity at 80° C./70° C.; and optionally    -   optionally a glucoamylase derived from Penicillium oxalicum.

The glucoamylase may optionally be substituted or combined with apullulanase preferably derived from Thermococcus litoralis orThermococcus hydrothermalis.

In a preferred embodiment the composition comprises

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   optionally a protease, preferably derived from Pyrococcus        furiosus or Thermoascus aurantiacus, having a thermostability        value of more than 20% determined as Relative Activity at 80°        C./70° C.;    -   optionally a glucoamylase derived from Penicillium oxalicum.

In a preferred embodiment the composition comprises

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182 and substitution N193F; and        optionally further one of the following set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   V59A+E129V+K177L+R179E+Q254S+M284V;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering).    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.        derived from Pyrococcus furiosus and/or Thermoascus aurantiacus;        and    -   optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 14        having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering).

In an embodiment the composition comprises:

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182+N193F; and further one of the        following set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   V59A+E129V+K177L+R179E+Q254S+M284V    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering).    -   a protease derived from Pyrococcus furiosus, preferably the one        shown in SEQ ID NO: 13 herein or SEQ ID NO: 29 herein;    -   a Penicillium oxalicum glucoamylase shown in SEQ ID NO: 14        having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering);

In an embodiment the invention relates to compositions comprising

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion        I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using        SEQ ID NO: 1 herein for numbering).    -   a protease derived from Pyrococcus furiosus, preferably the one        in SEQ ID NO: 13 herein or 29 herein;    -   a Penicillium oxalicum glucoamylase shown in SEQ ID NO: 14        having substitutions selected from the group of:    -   K79V+P11F+T65A+Q327F    -   K79V+P2N+P4F+P11F+T65A+Q327F (using SEQ ID NO: 14 for        numbering).

In an embodiment the Bacillus stearothermophilus alpha-amylase (SEQ IDNO: 1 herein), or a variant thereof, is the mature alpha-amylase orcorresponding mature alpha-amylases having at least 80% identity, atleast 90% identity, at least 95% identity at least 96% identity at least97% identity at least 99% identity to the SEQ ID NO: 1.

In an embodiment the Pyrococcus furiosus protease (SEQ ID NO: 13 hereinor SEQ ID NO: 20 herein) and/or Thermoascus aurantiacus protease (SEQ IDNO: 3), or a variant thereof, is the mature protease or correspondingmature protease having at least 80% identity, at least 90% identity, atleast 95% identity at least 96% identity at least 97% identity at least99% identity to the SEQ ID NO: 13 herein or SEQ ID NO: 29 herein, or SEQID NO: 3, respectively.

In an embodiment the Penicillium oxalicum glucoamylase (SEQ ID NO: 14herein), or a variant thereof, is the mature glucoamylase orcorresponding mature glucoamylase having at least 80% identity, at least90% identity, at least 95% identity at least 96% identity, at least 97%,at least 98% identity, or at least 99% identity to the SEQ ID NO: 14herein.

In an embodiment the carbohydrate-source generating enzyme, inparticular glucoamylase, is derived from a strain of Penicillium, suchas Penicillium oxalicum.

Materials & Methods Materials: Alpha-Amylase A (AAA):

Bacillus stearothermophilus alpha-amylase with the mutationsI181*+G182*+N193F truncated to 491 amino acids (SEQ ID NO: 1)

Alpha-Amylase 1407 (AA1407):

Bacillus stearothermophilus alpha-amylase with the mutationsI181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254Struncated to 491 amino acids (SEQ ID NO: 1)

Alpha-Amylase 369 (AA369): Bacillus stearothermophilus alpha-amylasewith the mutations:I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to491 amino acids (SEQ ID NO: 1);

Protease 196:

Metallo protease derived from Thermoascus aurantiacus CGMCC No. 0670disclosed as amino acids 1-177 in SEQ ID NO: 3 herein and amino acids1-177 in SEQ ID NO: in WO 2003/048353 with the following mutations:A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

Protease Pfu:

Protease derived from Pyrococcus furiosus purchased from Takara Bio(Japan) as Pfu Protease S (activity 10.5 mg/mL) and also shown in SEQ IDNO: 13 herein.

Protease Pfu2:

Protease derived from Pyrococcus furiosus shown in SEQ ID NO: 29 herein

Glucoamylase PO:

Mature part of the Penicillium oxalicum glucoamylase disclosed as SEQ IDNO: 2 in PCT/CN10/071753 published as WO 2011/127802 and shown in SEQ IDNO: 9 herein.

Glucoamylase PE001:

Variant of the Penicillium oxalicum glucoamylase having a K79Vsubstitution using the mature sequence shown in SEQ ID NO: 14 fornumbering.

Glucoamylase 493 (GA493):

Variant of Penicillium oxalicum glucoamylase variant PE001 furtherhaving the following substitutions: P11F+T65A+Q327F (using SEQ ID NO: 14for numbering).

Glucoamylase 498 (GA498):

Variant of Penicillium oxalicum glucoamylase variant PE001 furtherhaving the following substitutions: P2N+P4F+P11F+T65A+Q327F (using SEQID NO: 14 for numbering).

Glucoamylase BL:

Blend of Talaromyces emersonii glucoamylase disclosed in WO 99/28448 asSEQ ID NO: 7 and Trametes cingulata glucoamylase disclosed in WO06/069289 in a ratio of about 9:1.

Glucoamylase BL2:

Blend comprising Talaromyces emersonii glucoamylase disclosed inWO99/28448, Trametes cingulata glucoamylase disclosed in WO 06/69289,and Rhizomucor pusillus alpha-amylase with Aspergillus nigerglucoamylase linker and SBD disclosed as V039 in Table 5 in WO2006/069290 as side activities (ratio about 65:15:1).

Glucoamylase BL3:

Blend comprising Talaromyces emersonii glucoamylase disclosed inWO99/28448, Trametes cingulata glucoamylase disclosed in WO 06/69289,and Rhizomucor pusillus alpha-amylase with Aspergillus nigerglucoamylase linker and SBD disclosed as V039 in Table 5 in WO2006/069290 as side activities (ratio about 21:5:1).

Glucoamylase BL4:

Blend comprising Talaromyces emersonii glucoamylase disclosed inWO99/28448, Trametes cingulata glucoamylase disclosed in WO 06/69289,and Rhizomucor pusillus alpha-amylase with Aspergillus nigerglucoamylase linker and SBD disclosed as V039 in Table 5 in WO2006/069290 with the following substitutions: G128D+D143N (activityratio AGU:AGU:FAU(F): approx. 30:7:1).

Cellulolytic Composition A (CCA):

Cellulase composition from Trichoderma reesei sold as CELLUCLAST 1.5 L(Novozymes NS, Denmark)

Cellulolytic Composition B (CCB):

Cellulolytic composition derived from Trichoderma reesei comprisingGH61A polypeptide having cellulolytic enhancing activity derived from astrain of Penicillium emersonii (SEQ ID NO: 2 in WO 2011/041397 or SEQID NO: 23 herein), Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2in WO 2005/047499 SEQ ID NO: 22 herein) variant F100D, S283G, N456E,F512Y) disclosed in WO 2012/044915; Aspergillus fumigatus Cel7A CBH1disclosed as SEQ ID NO: 6 in WO2011/057140 (SEQ ID NO: 24 herein) andAspergillus fumigatus CBH II disclosed as SEQ ID NO: 18 in WO2011/057140 (SEQ ID NO: 25 herein).

Yeast:

RED STAR ETHANOL RED™ available from Red Star/Lesaffre, USA.

Substrate in Examples 18 and 19:

Ground corn and backset was obtained from a commercial plant in the USA.

Methods Identity:

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “identity”.

For purposes of the present invention the degree of identity between twoamino acid sequences, as well as the degree of identity between twonucleotide sequences, may be determined by the program “align” which isa Needleman-Wunsch alignment (i.e. a global alignment). The program isused for alignment of polypeptide, as well as nucleotide sequences. Thedefault scoring matrix BLOSUM50 is used for polypeptide alignments, andthe default identity matrix is used for nucleotide alignments. Thepenalty for the first residue of a gap is −12 for polypeptides and −16for nucleotides. The penalties for further residues of a gap are −2 forpolypeptides, and −4 for nucleotides.

“Align” is part of the FASTA package version v20u6 (see W. R. Pearsonand D. J. Lipman (1988), “Improved Tools for Biological SequenceAnalysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid andSensitive Sequence Comparison with FASTP and FASTA,” Methods inEnzymology 183:63-98). FASTA protein alignments use the Smith-Watermanalgorithm with no limitation on gap size (see “Smith-Watermanalgorithm”, T. F. Smith and M. S. Waterman (1981) J. Mol. Biol.147:195-197).

Protease Assays AZCL-Casein Assay

A solution of 0.2% of the blue substrate AZCL-casein is suspended inBorax/NaH₂PO₄ buffer pH9 while stirring. The solution is distributedwhile stirring to microtiter plate (100 microL to each well), 30 microLenzyme sample is added and the plates are incubated in an EppendorfThermomixer for 30 minutes at 45° C. and 600 rpm. Denatured enzymesample (100° C. boiling for 20 min) is used as a blank. After incubationthe reaction is stopped by transferring the microtiter plate onto iceand the coloured solution is separated from the solid by centrifugationat 3000 rpm for 5 minutes at 4° C. 60 microL of supernatant istransferred to a microtiter plate and the absorbance at 595 nm ismeasured using a BioRad Microplate Reader.

pNA-Assay

50 microL protease-containing sample is added to a microtiter plate andthe assay is started by adding 100 microL 1 mM pNA substrate (5 mgdissolved in 100 microL DMSO and further diluted to 10 mL withBorax/NaH₂PO₄ buffer pH 9.0). The increase in OD₄₀₅ at room temperatureis monitored as a measure of the protease activity.

Glucoamylase Activity (AGU)

Glucoamylase activity may be measured in Glucoamylase Units (AGU).

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesEnzyme working range: 0.5-4.0 AGU/mL

Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer:phosphate 0.12M; 0.15M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37°C. ± 1 Reaction time: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes NS, Denmark, which folderis hereby included by reference.

Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid FungalAlpha-amylase Units), which are determined relative to an enzymestandard. 1 AFAU is defined as the amount of enzyme which degrades 5.260mg starch dry matter per hour under the below mentioned standardconditions.

Acid alpha-amylase, an endo-alpha-amylase(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzesalpha-1,4-glucosidic bonds in the inner regions of the starch moleculeto form dextrins and oligosaccharides with different chain lengths. Theintensity of color formed with iodine is directly proportional to theconcentration of starch. Amylase activity is determined using reversecolorimetry as a reduction in the concentration of starch under thespecified analytical conditions.

Standard Conditions/Reaction Conditions:

-   -   Substrate: Soluble starch, approx. 0.17 g/L    -   Buffer: Citrate, approx. 0.03 M    -   Iodine (I2): 0.03 g/L    -   CaCl2: 1.85 mM    -   pH: 2.50±0.05    -   Incubation temperature: 40° C.    -   Reaction time: 23 seconds    -   Wavelength: 590 nm    -   Enzyme concentration: 0.025 AFAU/mL    -   Enzyme working range: 0.01-0.04 AFAU/mL

A folder EB-SM-0259.02/01 describing this analytical method in moredetail is available upon request to Novozymes NS, Denmark, which folderis hereby included by reference.

Alpha-Amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch assubstrate. This method is based on the break-down of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution. Initially, ablackish-blue color is formed, but during the break-down of the starchthe blue color gets weaker and gradually turns into a reddish-brown,which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance MerckAmylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in moredetail is available upon request to Novozymes NS, Denmark, which folderis hereby included by reference.

Determination of FAU(F)

FAU(F) Fungal Alpha-Amylase Units (Fungamyl) is measured relative to anenzyme standard of a declared strength.

Reaction conditions Temperature 37° C. pH 7.15 Wavelength 405 nmReaction time 5 min Measuring time 2 min

A folder (EB-SM-0216.02) describing this standard method in more detailis available on request from Novozymes NS, Denmark, which folder ishereby included by reference.

Determination of Pullulanase Activity (NPUN)

Endo-pullulanase activity in NPUN is measured relative to a Novozymespullulanase standard. One pullulanase unit (NPUN) is defined as theamount of enzyme that releases 1 micro mol glucose per minute under thestandard conditions (0.7% red pullulan (Megazyme), pH 5, 40° C., 20minutes). The activity is measured in NPUN/ml using red pullulan.

1 mL diluted sample or standard is incubated at 40° C. for 2 minutes.0.5 mL 2% red pullulan, 0.5 M KCl, 50 mM citric acid, pH 5 are added andmixed. The tubes are incubated at 40° C. for 20 minutes and stopped byadding 2.5 ml 80% ethanol. The tubes are left standing at roomtemperature for 10-60 minutes followed by centrifugation 10 minutes at4000 rpm. OD of the supernatants is then measured at 510 nm and theactivity calculated using a standard curve.

The present invention is described in further detail in the followingexamples which are offered to illustrate the present invention, but notin any way intended to limit the scope of the invention as claimed. Allreferences cited herein are specifically incorporated by reference forthat which is described therein.

EXAMPLES Example 1 Stability of Alpha-Amylase Variants

The stability of a reference alpha-amylase (Bacillus stearothermophilusalpha-amylase with the mutations I181*+G182*+N193F truncated to 491amino acids (SEQ ID NO: 1 numbering)) and alpha-amylase variants thereofwas determined by incubating the reference alpha-amylase and variants atpH 4.5 and 5.5 and temperatures of 75° C. and 85° C. with 0.12 mM CaCl₂followed by residual activity determination using the EnzChek® substrate(EnzChek® Ultra Amylase assay kit, E33651, Molecular Probes).

Purified enzyme samples were diluted to working concentrations of 0.5and 1 or 5 and 10 ppm (micrograms/ml) in enzyme dilution buffer (10 mMacetate, 0.01% Triton X100, 0.12 mM CaCl₂, pH 5.0). Twenty microlitersenzyme sample was transferred to 48-well PCR MTP and 180 microlitersstability buffer (150 mM acetate, 150 mM MES, 0.01% Triton X100, 0.12 mMCaCl₂, pH 4.5 or 5.5) was added to each well and mixed. The assay wasperformed using two concentrations of enzyme in duplicates. Beforeincubation at 75° C. or 85° C., 20 microliters was withdrawn and storedon ice as control samples. Incubation was performed in a PCR machine at75° C. and 85° C. After incubation samples were diluted to 15 ng/mL inresidual activity buffer (100 mM Acetate, 0.01% Triton X100, 0.12 mMCaCl₂, pH 5.5) and 25 microliters diluted enzyme was transferred toblack 384-MTP. Residual activity was determined using the EnzCheksubstrate by adding 25 microliters substrate solution (100micrograms/ml) to each well. Fluorescence was determined every minutefor 15 minutes using excitation filter at 485-P nm and emission filterat 555 nm (fluorescence reader is Polarstar, BMG). The residual activitywas normalized to control samples for each setup.

Assuming logarithmic decay half life time (T½ (min)) was calculatedusing the equation: T½(min)=T(min)*LN(0.5)/LN(% RA/100), where T isassay incubation time in minutes, and % RA is % residual activitydetermined in assay.

Using this assay setup the half life time was determined for thereference alpha-amylase and variant thereof as shown in Table 1.

TABLE 1 T½ (min) T½ (min) T½ (min) (pH 4.5, 75° C., (pH 4.5, 85° C., (pH5.5, 85° C., Mutations 0.12 mM CaCl₂) 0.12 mM CaCl₂) 0.12 mM CaCl₂)Reference Alpha-Amylase A 21 4 111 Reference Alpha-Amylase A with 32 6301 the substitution V59A Reference Alpha-Amylase A with 28 5 230 thesubstitution V59E Reference Alpha-Amylase A with 28 5 210 thesubstitution V59I Reference Alpha-Amylase A with 30 6 250 thesubstitution V59Q Reference Alpha-Amylase A with 149 22 ND thesubstitutions V59A + Q89R + G112D + E129V + K177L + R179E + K220P +N224L + Q254S Reference Alpha-Amylase A with >180 28 ND thesubstitutions V59A + Q89R + E129V + K177L + R179E + H208Y + K220P +N224L + Q254S Reference Alpha-Amylase A with 112 16 ND the substitutionsV59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + D269E +D281N Reference Alpha-Amylase A with 168 21 ND the substitutions V59A +Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + I270L ReferenceAlpha-Amylase A with >180 24 ND the substitutions V59A + Q89R + E129V +K177L + R179E + K220P + N224L + Q254S + H274K Reference Alpha-Amylase Awith 91 15 ND the substitutions V59A + Q89R + E129V + K177L + R179E +K220P + N224L + Q254S + Y276F Reference Alpha-Amylase A with 141 41 NDthe substitutions V59A + E129V + R157Y + K177L + R179E + K220P + N224L +S242Q + Q254S Reference Alpha-Amylase A with >180 62 ND thesubstitutions V59A + E129V + K177L + R179E + H208Y + K220P + N224L +S242Q + Q254S Reference Alpha-Amylase A with >180 49 >480 thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S Reference Alpha-Amylase A with >180 53 ND the substitutions V59A +E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + H274K ReferenceAlpha-Amylase A with >180 57 ND the substitutions V59A + E129V + K177L +R179E + K220P + N224L + S242Q + Q254S + Y276F Reference Alpha-Amylase Awith >180 37 ND the substitutions V59A + E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + D281N Reference Alpha-Amylase A with >180 51 NDthe substitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + M284T Reference Alpha-Amylase A with >180 45 ND thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + G416V Reference Alpha-Amylase A with 143 21 >480 thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + Q254SReference Alpha-Amylase A with >180 22 ND the substitutions V59A +E129V + K177L + R179E + K220P + N224L + Q254S + M284T ReferenceAlpha-Amylase A with >180 38 ND the substitutions A91L + M96I + E129V +K177L + R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith 57 11 402 the substitutions E129V + K177L + R179E ReferenceAlpha-Amylase A with 174 44 >480 the substitutions E129V + K177L +R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith >180 49 >480 the substitutions E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + Y276F + L427M Reference Alpha-Amylase Awith >180 49 >480 the substitutions E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + M284T Reference Alpha-Amylase A with 177 36 >480the substitutions E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + N376* + I377* Reference Alpha-Amylase A with 94 13 >480 thesubstitutions E129V + K177L + R179E + K220P + N224L + Q254S ReferenceAlpha-Amylase A with 129 24 >480 the substitutions E129V + K177L +R179E + K220P + N224L + Q254S + M284T Reference Alpha-Amylase A with 14830 >480 the substitutions E129V + K177L + R179E + S242Q ReferenceAlpha-Amylase A with 78 9 >480 the substitutions E129V + K177L + R179VReference Alpha-Amylase A with 178 31 >480 the substitutions E129V +K177L + R179V + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith 66 17 >480 the substitutions K220P + N224L + S242Q + Q254SReference Alpha-Amylase A with 30 6 159 the substitutions K220P +N224L + Q254S Reference Alpha-Amylase A with 35 7 278 the substitutionM284T Reference Alpha-Amylase A with 59 13 ND the substitutions M284V NDnot determined

The results demonstrate that the alpha-amylase variants have asignificantly greater half-life and stability than the referencealpha-amylase.

Example 2 Preparation of Protease Variants and Test of Thermostability

Strains and Plasmids

E. coli DH12S (available from Gibco BRL) was used for yeast plasmidrescue. pJTP000 is a S. cerevisiae and E. coli shuttle vector under thecontrol of TPI promoter, constructed from pJC039 described in WO01/92502, in which the Thermoascus aurantiacus M35 protease gene (WO03048353) has been inserted.

Saccharomyces cerevisiae YNG318 competent cells: MATa Dpep4[cir+]ura3-52, leu2-D2, his 4-539 was used for protease variants expression.It is described in J. Biol. Chem. 272 (15), pp 9720-9727, 1997.

Media and Substrates 10× Basal Solution:

Yeast nitrogen base w/o amino acids (DIFCO) 66.8 g/l, succinate 100 g/l,NaOH 60 g/l.

SC-Glucose:

20% glucose (i.e., a final concentration of 2%=2 g/100 ml)) 100 ml/l, 5%threonine 4 ml/l, 1% tryptophan 10 ml/l, 20% casamino acids 25 ml/l, 10×basal solution 100 ml/l. The solution is sterilized using a filter of apore size of 0.20 micrometer. Agar (2%) and H₂O (approx. 761 ml) isautoclaved together, and the separately sterilized SC-glucose solutionis added to the agar solution.

YPD:

Bacto peptone 20 g/l, yeast extract 10 g/l, 20% glucose 100 ml/l.

YPD+Zn:

YPD+0.25 mM ZnSO₄

PEG/LiAc Solution:

40% PEG4000 50 ml, 5 M Lithium Acetate 1 ml.

96 Well Zein Micro Titre Plate:

Each well contains 200 microL of 0.05-0.1% of zein (Sigma), 0.25 mMZnSO₄ and 1% of agar in 20 mM sodium acetate buffer, pH 4.5.

DNA Manipulations

Unless otherwise stated, DNA manipulations and transformations wereperformed using standard methods of molecular biology as described inSambrook et al. (1989) Molecular cloning: A laboratory manual, ColdSpring Harbor lab. Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.) “Current protocols in Molecular Biology”, John Wiley and Sons,1995; Harwood, C. R. and Cutting, S. M. (Eds.).

Yeast Transformation

Yeast transformation was performed using the lithium acetate method. 0.5microL of vector (digested by restriction endonucleases) and 1 microL ofPCR fragments is mixed. The DNA mixture, 100 microL of YNG318 competentcells, and 10 microL of YEAST MAKER carrier DNA (Clontech) is added to a12 ml polypropylene tube (Falcon 2059). Add 0.6 ml PEG/LiAc solution andmix gently. Incubate for 30 min at 30° C., and 200 rpm followed by 30min at 42° C. (heat shock). Transfer to an eppendorf tube and centrifugefor 5 sec. Remove the supernatant and resolve in 3 ml of YPD. Incubatethe cell suspension for 45 min at 200 rpm at 30° C. Pour the suspensionto SC-glucose plates and incubate 30° C. for 3 days to grow colonies.Yeast total DNA are extracted by Zymoprep Yeast Plasmid Miniprep Kit(ZYMO research).

DNA Sequencing

E. coli transformation for DNA sequencing was carried out byelectroporation (BIO-RAD Gene Pulser). DNA Plasmids were prepared byalkaline method (Molecular Cloning, Cold Spring Harbor) or with theQiagen® Plasmid Kit. DNA fragments were recovered from agarose gel bythe Qiagen gel extraction Kit. PCR was performed using a PTC-200 DNAEngine. The ABI PRISM™ 310 Genetic Analyzer was used for determinationof all DNA sequences.

Construction of Protease Expression Vector

The Thermoascus M35 protease gene was amplified with the primer pairProt F (SEQ ID NO: 4) and Prot R (SEQ ID NO: 5). The resulting PCRfragments were introduced into S. cerevisiae YNG318 together with thepJC039 vector (described in WO 2001/92502) digested with restrictionenzymes to remove the Humicola insolens cutinase gene.

The Plasmid in yeast clones on SC-glucose plates was recovered toconfirm the internal sequence and termed as pJTP001.

Construction of Yeast Library and Site-Directed Variants

Library in yeast and site-directed variants were constructed by SOE PCRmethod (Splicing by Overlap Extension, see “PCR: A practical approach”,p. 207-209, Oxford University press, eds. McPherson, Quirke, Taylor),followed by yeast in vivo recombination.

General Primers for Amplification and Sequencing

The primers AM34 (SEQ ID NO:5) and AM35 (SEQ ID NO:6) were used to makeDNA fragments containing any mutated fragments by the SOE methodtogether with degenerated primers (AM34+Reverse primer and AM35+forwardprimer) or just to amplify a whole protease gene (AM34+AM35).

PCR reaction system: Conditions: 48.5 microL H₂O 1 94° C. 2 min 2 beadspuRe Taq Ready-To-Go PCR (Amersham 2 94° C. 30 sec Biosciences) 3 55° C.30 sec 0.5 micro L X 2 100 pmole/microL of primers 4 72° C. 90 sec 0.5microL template DNA 2-4 25 cycles 5 72° C. 10 min

DNA fragments were recovered from agarose gel by the Qiagen gelextraction Kit. The resulting purified fragments were mixed with thevector digest. The mixed solution was introduced into Saccharomycescerevisiae to construct libraries or site-directed variants by in vivorecombination.

Relative Activity Assay

Yeast clones on SC-glucose were inoculated to a well of a 96-well microtitre plate containing YPD+Zn medium and cultivated at 28° C. for 3days. The culture supernatants were applied to a 96-well zein microtiter plate and incubated at at least 2 temperatures (ex. 60° C. and 65°C., 70° C. and 75° C., 70° C. and 80° C.) for more than 4 hours orovernight. The turbidity of zein in the plate was measured as A630 andthe relative activity (higher/lower temperatures) was determined as anindicator of thermoactivity improvement. The clones with higher relativeactivity than the parental variant were selected and the sequence wasdetermined.

Remaining Activity Assay

Yeast clones on SC-glucose were inoculated to a well of a 96-well microtitre plate and cultivated at 28° C. for 3 days. Protease activity wasmeasured at 65° C. using azo-casein (Megazyme) after incubating theculture supernatant in 20 mM sodium acetate buffer, pH 4.5, for 10 minat a certain temperature (80° C. or 84° C. with 4° C. as a reference) todetermine the remaining activity. The clones with higher remainingactivity than the parental variant were selected and the sequence wasdetermined.

Azo-Casein Assay

20 microL of samples were mixed with 150 microL of substrate solution (4ml of 12.5% azo-casein in ethanol in 96 ml of 20 mM sodium acetate, pH4.5, containing 0.01% triton-100 and 0.25 mM ZnSO₄) and incubated for 4hours or longer.

After adding 20 microL/well of 100% trichloroacetic acid (TCA) solution,the plate was centrifuge and 100 microL of supernatants were pipette outto measure A440.

Expression of Protease Variants in Aspergillus oryzae

The constructs comprising the protease variant genes were used toconstruct expression vectors for Aspergillus. The Aspergillus expressionvectors consist of an expression cassette based on the Aspergillus nigerneutral amylase II promoter fused to the Aspergillus nidulans triosephosphate isomerase non translated leader sequence (Pna2/tpi) and theAspergillus niger amyloglucosidase terminator (Tamg). Also present onthe plasmid was the Aspergillus selective marker amdS from Aspergillusnidulans enabling growth on acetamide as sole nitrogen source. Theexpression plasmids for protease variants were transformed intoAspergillus as described in Lassen et al. (2001), Appl. Environ.Microbiol. 67, 4701-4707. For each of the constructs 10-20 strains wereisolated, purified and cultivated in shake flasks.

Purification of Expressed Variants

-   1. Adjust pH of the 0.22 μm filtered fermentation sample to 4.0.-   2. Put the sample on an ice bath with magnetic stirring. Add    (NH4)2SO4 in small aliquots (corresponding to approx. 2.0-2.2 M    (NH4)2SO4 not taking the volume increase into account when adding    the compound).-   3. After the final addition of (NH4)2SO4, incubate the sample on the    ice bath with gentle magnetic stirring for min. 45 min.-   4. Centrifugation: Hitachi himac CR20G High-Speed Refrigerated    Centrifuge equipped with R20A2 rotor head, 5° C., 20,000 rpm, 30    min.-   5. Dissolve the formed precipitate in 200 ml 50 mM Na-acetate pH    4.0.-   6. Filter the sample by vacuum suction using a 0.22 μm PES PLUS    membrane (IWAKI).-   7. Desalt/buffer-exchange the sample to 50 mM Na-acetate pH 4.0    using ultrafiltration (Vivacell 250 from Vivascience equipped with 5    kDa MWCO PES membrane) overnight in a cold room. Dilute the    retentate sample to 200 ml using 50 mM Na-acetate pH 4.0. The    conductivity of sample is preferably less than 5 mS/cm.-   8. Load the sample onto a cation-exchange column equilibrated with    50 mM Na-acetate pH 4.0. Wash unbound sample out of the column using    3 column volumes of binding buffer (50 mM Na-acetate pH 4.0), and    elute the sample using a linear gradient, 0-100% elution buffer (50    mM Na-acetate+1 M NaCl pH 4.0) in 10 column volumes.-   9. The collected fractions are assayed by an endo-protease assay    (cf. below) followed by standard SDS-PAGE (reducing conditions) on    selected fractions. Fractions are pooled based on the endo-protease    assay and SDS-PAGE.

Endo-Protease Assay

-   1. Protazyme OL tablet/5 ml 250 mM Na-acetate pH 5.0 is dissolved by    magnetic stirring (substrate: endo-protease Protazyme AK tablet from    Megazyme—cat. # PRAK 11/08).-   2. With stirring, 250 microL of substrate solution is transferred to    a 1.5 ml Eppendorf tube.-   3. 25 microL of sample is added to each tube (blank is sample    buffer).-   4. The tubes are incubated on a Thermomixer with shaking (1000 rpm)    at 50° C. for 15 minutes.-   5. 250 microL of 1 M NaOH is added to each tube, followed by    vortexing.-   6. Centrifugation for 3 min. at 16,100×G and 25° C.-   7. 200 microL of the supernatant is transferred to a MTP, and the    absorbance at 590 nm is recorded.

Results

TABLE 2 Relative activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 2. Relative activity VariantSubstitution(s) 65° C./60° C. WT none 31% JTP004 S87P 45% JTP005 A112P43% JTP008 R2P 71% JTP009 D79K 69% JTP010 D79L 75% JTP011 D79M 73%JTP012 D79L/S87P 86% JTP013 D79L/S87P/A112P 90% JTP014 D79L/S87P/A112P88% JTP016 A73C 52% JTP019 A126V 69% JTP021 M152R 59%

TABLE 3 Relative activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 2. Relative activity Substitution(s) and/70° C./ 75° C./ 75° C./ Variant or deletion (S) 65° C. 65° C. 70° C. WTnone 59% 17% JTP036 D79L/S87P/D142L 73% 73% JTP040 T54R/D79L/S87P 71%JTP042 Q53K/D79L/S87P/I173V 108%  JTP043 Q53R/D79L/S87P 80% JTP045S41R/D79L/S87P 82% JTP046 D79L/S87P/Q158W 96% JTP047 D79L/S87P/S157K 85%JTP048 D79L/S87P/D104R 88% JTP050 D79L/S87P/A112P/D142L 88% JTP051S41R/D79L/S87P/A112P/D142L 102% JTP052 D79L/S87P/A112P/D142L/S157K 111%JTP053 S41R/D79L/S87P/A112P/D142L/ 113% S157K JTP054 ΔS5/D79L/S87P  92%JTP055 ΔG8/D79L/S87P  95% JTP059 C6R/D79L/S87P  92% JTP061T46R/D79L/S87P 111% JTP063 S49R/D79L/S87P  94% JTP064 D79L/S87P/N88R 92% JTP068 D79L/S87P/T114P  99% JTP069 D79L/S87P/S115R 103% JTP071D79L/S87P/T116V 105% JTP072 N26R/D79L/S87P 92% JTP077A27K/D79L/S87P/A112P/D142L 106%  JTP078 A27V/D79L/S87P/A112P/D142L 100% JTP079 A27G/D79L/S87P/A112P/D142L 104% 

TABLE 4 Relative activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 2. Relative activity RemainingSubstitution(s) and/ 75° C./ activity Variant or deletion(s) 65° C. 80°C. 84° C. JTP082 ΔS5/D79L/S87P/A112P/D142L 129% 53% JTP083T46R/D79L/S87P/A112P/D142L 126% JTP088 Y43F/D79L/S87P/A112P/D142L 119%JTP090 D79L/S87P/A112P/T124L/D142L 141% JTP091D79L/S87P/A112P/T124V/D142L 154% 43% JTP092 ΔS5/N26R/D79L/S87P/A112P/60% D142L JTP095 N26R/T46R/D79L/S87P/A112P/ 62% D142L JTP096T46R/D79L/S87P/T116V/D142L 67% JTP099 D79L/P81R/S87P/A112P/D142L 80%JTP101 A27K/D79L/S87P/A112P/T124V/ 81% D142L JTP116D79L/Y82F/S87P/A112P/T124V/ 59% D142L JTP117 D79L/Y82F/S87P/A112P/T124V/94% D142L JTP127 D79L/S87P/A112P/T124V/A126V/ 53% D142L

TABLE 5 Relative activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 2. Relative activity 75° C./ 80° C./ 85°C./ Variant Substitutions 70° C. 70° C. 70° C. JTP050 D79L S87P A112PD142L 55% 23%  9% JTP134 D79L Y82F S87P A112P D142L 40% JTP135 S38T D79LS87P A112P A126V 62% D142L JTP136 D79L Y82F S87P A112P A126V 59% D142LJTP137 A27K D79L S87P A112P A126V 54% D142L JTP140 D79L S87P N98C A112PG135C 81% D142L JTP141 D79L S87P A112P D142L T141C 68% M161C JTP143 S36PD79L S87P A112P D142L 69% JTP144 A37P D79L S87P A112P D142L 57% JTP145S49P D79L S87P A112P D142L 82% 59% JTP146 S50P D79L S87P A112P D142L 83%63% JTP148 D79L S87P D104P A112P D142L 76% 64% JTP161 D79L Y82F S87GA112P D142L 30% 12% JTP180 S70V D79L Y82F S87G Y97W 52% A112P D142LJTP181 D79L Y82F S87G Y97W D104P 45% A112P D142L JTP187 S70V D79L Y82FS87G A112P 45% D142L JTP188 D79L Y82F S87G D104P A112P 43% D142L JTP189D79L Y82F S87G A112P A126V 46% D142L JTP193 Y82F S87G S70V D79L D104P15% A112P D142L JTP194 Y82F S87G D79L D104P A112P 22% A126V D142L JTP196A27K D79L Y82F S87G D104P 18% A112P A126V D142L

TABLE 5 Relative activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 2. Relative activity Variant Substitutions75° C./70° C. 80° C./70° C. JTP196 A27K D79L Y82F 102% 55% S87G D104PA112P A126V D142L JTP210 A27K Y82F S87G 107% 36% D104P A112P A126V D142LJTP211 A27K D79L Y82F  94% 44% D104P A112P A126V D142L JTP213 A27K Y82FD104P 103% 37% A112P A126V D142L

Example 3 Temperature Profile of Selected Variants Using PurifiedEnzymes

Selected variants showing good thermo-stability were purified and thepurified enzymes were used in a zein-BCA assay as described below. Theremaining protease activity was determined at 60° C. after incubation ofthe enzyme at elevated temperatures as indicated for 60 min.

Zein-BCA Assay:

Zein-BCA assay was performed to detect soluble protein quantificationreleased from zein by variant proteases at various temperatures.

Protocol:

-   1) Mix 10 ul of 10 ug/ml enzyme solutions and 100 ul of 0.025% zein    solution in a micro titer plate (MTP).-   2) Incubate at various temperatures for 60 min.-   3) Add 10 ul of 100% trichloroacetic acid (TCA) solution.-   4) Centrifuge MTP at 3500 rpm for 5 min.-   5) Take out 15 ul to a new MTP containing 100 ul of BCA assay    solution (Pierce Cat#:23225, BCA Protein Assay Kit).-   6) Incubate for 30 min. at 60° C.-   7) Measure A562.    The results are shown in Table 6. All of the tested variants showed    an improved thermo-stability as compared to the wt protease.

TABLE 6 Zein-BCA assay Sample incubated 60 min at indicated temperatures(° C.) (μg/ml Bovine serum albumin equivalent peptide released) WT/ 60°70° 75° 80° 85° 90° 95° Variant C. C. C. C. C. C. C. WT 94 103 107 93 5838 JTP050 86 101 107 107 104 63 36 JTP077 82 94 104 105 99 56 31 JTP18871 83 86 93 100 75 53 JTP196 87 99 103 106 117 90 38

Example 4 Characterization of Penicillium oxalicum Glucoamylase

The Penicillium oxalicum glucoamylase is disclosed in SEQ ID NO: 9herein.

Substrate.

Substrate: 1% soluble starch (Sigma S-9765) in deionized water

Reaction buffer: 0.1 M Acetate buffer at pH 5.3Glucose concentration determination kit: Wako glucose assay kit(LabAssay glucose, WAKO, Cat#298-65701).

Reaction Condition.

20 microL soluble starch and 50 microL acetate buffer at pH 5.3 weremixed. 30 microL enzyme solution (50 micro g enzyme protein/ml) wasadded to a final volume of 100 microL followed by incubation at 37° C.for 15 min.

The glucose concentration was determined by Wako kits.

All the work carried out in parallel.

Temperature Optimum.

To assess the temperature optimum of the Penicillium oxalicumglucoamylase the “Reaction condition”-assay described above wasperformed at 20, 30, 40, 50, 60, 70, 80, 85, 90 and 95° C. The resultsare shown in Table 7.

TABLE 7 Temperature optimum Temperature (° C.) 20 30 40 50 60 70 80 8590 95 Relative 63.6 71.7 86.4 99.4 94.6 100.0 92.9 92.5 82.7 82.8activity (%)From the results it can be seen that the optimal temperature forPenicillium oxalicum glucoamylase at the given conditions is between 50°C. and 70° C. and the glucoamylase maintains more than 80% activity at95° C.

Heat Stability.

To assess the heat stability of the Penicillium oxalicum glucoamylasethe Reaction condition assay was modified in that the enzyme solutionand acetate buffer was preincubated for 15 min at 20, 30, 40, 50, 60,70, 75, 80, 85, 90 and 95° C. Following the incubation 20 microL ofstarch was added to the solution and the assay was performed asdescribed above.

The results are shown in Table 8.

TABLE 8 Heat stability Temperature (° C.) 20 30 40 50 60 70 80 85 90 95Relative 91.0 92.9 88.1 100.0 96.9 86.0 34.8 36.0 34.2 34.8 activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylaseis stable up to 70° C. after preincubation for 15 min in that itmaintains more than 80% activity.

pH Optimum.

To assess the pH optimum of the Penicillium oxalicum glucoamylase theReaction condition assay described above was performed at pH 2.0, 3.0,3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 and 11.0. Instead of usingthe acetate buffer described in the Reaction condition assay thefollowing buffer was used 100 mM Succinic acid, HEPES, CHES, CAPSO, 1 mMCaCl₂, 150 mM KCl, 0.01% Triton X-100, pH adjusted to 2.0, 3.0, 3.5,4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 or 11.0 with HCl or NaOH.

The results are shown in Table 9.

TABLE 9 pH optimum pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 10.0 11.0Relative 71.4 78.6 77.0 91.2 84.2 100.0 55.5 66.7 30.9 17.8 15.9 16.1activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylaseat the given conditions has the highest activity at pH 5.0. ThePenicillium oxalicum glucoamylase is active in a broad pH range in theit maintains more than 50% activity from pH 2 to 7.

pH Stability.

To assess the heat stability of the Penicillium oxalicum glucoamylasethe Reaction condition assay was modified in that the enzyme solution(50 micro g/mL) was preincubated for 20 hours in buffers with pH 2.0,3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 and 11.0 using thebuffers described under pH optimum. After preincubation, 20 microLsoluble starch to a final volume of 100 microL was added to the solutionand the assay was performed as described above.

The results are shown in Table 10.

TABLE 10 pH stability pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 10.011.0 Relative 17.4 98.0 98.0 103.2 100.0 93.4 71.2 90.7 58.7 17.4 17.017.2 activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylase,is stable from pH 3 to pH 7 after preincubation for 20 hours and itdecreases its activity at pH 8.

Example 5 Thermostability of Protease Pfu

The thermostability of the Pyrococcus furiosus protease (Pfu S)purchased from Takara Bio Inc, (Japan) was tested using the same methodsas in Example 2. It was found that the thermostability (RelativeActivity) was 110% at (80° C./70° C.) and 103% (90° C./70° C.) at pH4.5.

Example 6 Cloning of Penicillium oxalicum Strain Glucoamylase Gene

Preparation of Penicillium oxalicum Strain cDNA.

The cDNA was synthesized by following the instruction of 3′ RapidAmplification of cDNA End System (Invitrogen Corp., Carlsbad, Calif.,USA).

Cloning of Penicillium oxalicum Strain Glucoamylase Gene.

The Penicillium oxalicum glucoamylase gene was cloned using theoligonucleotide primer shown below designed to amplify the glucoamylasegene from 5′ end.

Sense primer: (SEQ ID NO: 15) 5′-ATGCGTCTCACTCTATTATCAGGTG-3′

The full length gene was amplified by PCR with Sense primer and AUAP(supplied by 3′ Rapid Amplification of cDNA End System) by usingPlatinum HIFI Taq DNA polymerase (Invitrogen Corp., Carlsbad, Calif.,USA). The amplification reaction was composed of 5 μl of 10×PCR buffer,2 μl of 25 mM MgCl₂, 1 μl of 10 mM dNTP, 1 μl of 10 uM Sense primer, 1μl of 10 uM AUAP, 2 μl of the first strand cDNA, 0.5 μl of HIFI Taq, and37.5 μl of deionized water. The PCR program was: 94° C., 3 mins; 10cycles of 94° C. for 40 secs, 60° C. 40 secs with 1° C. decrease percycle, 68° C. for 2 min; 25 cycles of 94° C. for 40 secs, 50° C. for 40secs, 68° C. for 2 min; final extension at 68° C. for 10 mins.

The obtained PCR fragment was cloned into pGEM-T vector (PromegaCorporation, Madison, Wis., USA) using a pGEM-T Vector System (PromegaCorporation, Madison, Wis., USA) to generate plasmid AMG 1. Theglucoamylase gene inserted in the plasmid AMG 1 was sequencingconfirmed. E. coli strain TOP10 containing plasmid AMG 1 (designatedNN059173), was deposited with the Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH (DSMZ) on Nov. 23, 2009, and assigned accessionnumber as DSM 23123.

Example 7 Expression of Cloned Penicillium oxalicum Glucoamylase

The Penicillium oxalicum glucoamylase gene was re-cloned from theplasmid AMG 1 into an Aspergillus expression vector by PCR using twocloning primer F and primer R shown below, which were designed based onthe known sequence and added tags for direct cloning by IN-FUSION™strategy.

Primer F: (SEQ ID NO: 16) 5′ ACACAACTGGGGATCCACCATGCGTCTCACTCTATTATCPrimer R: (SEQ ID NO: 17) 5′ AGATCTCGAGAAGCTTAAAACTGCCACACGTCGTTGG

A PCR reaction was performed with plasmid AMG 1 in order to amplify thefull-length gene. The PCR reaction was composed of 40 μg of the plasmidAMG 1 DNA, 1 μl of each primer (100 μM); 12.5 μl of 2× ExtensorHi-Fidelity master mix (Extensor Hi-Fidelity Master Mix, ABgene, UnitedKingdom), and 9.5 μl of PCR-grade water. The PCR reaction was performedusing a DYAD PCR machine (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA) programmed for 2 minutes at 94° C. followed by a 25 cycles of 94°C. for 15 seconds, 50° C. for 30 seconds, and 72° C. for 1 minute; andthen 10 minutes at 72° C.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 1×TAE buffer where an approximately 1.9 kb PCR product band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit (GE Healthcare, United Kingdom) according tomanufacturer's instructions. DNA corresponding to the Penicilliumoxalicum glucoamylase gene was cloned into an Aspergillus expressionvector linearized with BamHI and HindIII, using an IN-FUSION™ Dry-DownPCR Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) according tothe manufacturer's instructions. The linearized vector construction isas described in WO 2005/042735 A1.

A 2 μl volume of the ligation mixture was used to transform 25 μl ofFusion Blue E. coli cells (included in the IN-FUSION™ Dry-Down PCRCloning Kit). After a heat shock at 42° C. for 45 sec, and chilling onice, 250 μl of SOC medium was added, and the cells were incubated at 37°C. at 225 rpm for 90 min before being plated out on LB agar platescontaining 50 μg of ampicillin per ml, and cultivated overnight at 37°C. Selected colonies were inoculated in 3 ml of LB medium supplementedwith 50 μg of ampicillin per ml and incubated at 37° C. at 225 rpmovernight. Plasmid DNA from the selected colonies was purified usingMini JETSTAR (Genomed, Germany) according to the manufacturer'sinstructions. Penicillium oxalicum glucoamylase gene sequence wasverified by Sanger sequencing before heterologous expression. One of theplasmids was selected for further expression, and was named XYZXYZ1471-4.

Protoplasts of Aspergillus niger MBin118 were prepared as described inWO 95/02043. One hundred μl of protoplast suspension were mixed with 2.5μg of the XYZ1471-4 plasmid and 250 microliters of 60% PEG 4000(Applichem) (polyethylene glycol, molecular weight 4,000), 10 mM CaCl₂,and 10 mM Tris-HCl pH 7.5 were added and gently mixed. The mixture wasincubated at 37° C. for 30 minutes and the protoplasts were mixed with6% low melting agarose (Biowhittaker Molecular Applications) in COVEsucrose (Cove, 1996, Biochim. Biophys. Acta 133:51-56) (1M) platessupplemented with 10 mM acetamide and 15 mM CsCl and added as a toplayer on COVE sucrose (1M) plates supplemented with 10 mM acetamide and15 mM CsCl for transformants selection (4 ml topagar per plate). Afterincubation for 5 days at 37° C. spores of sixteen transformants werepicked up and seed on 750 μl YP-2% Maltose medium in 96 deepwell MTplates. After 5 days of stationary cultivation at 30° C., 10 μl of theculture-broth from each well was analyzed on a SDS-PAGE (Sodium dodecylsulfate-polyacrylamide gel electrophoresis) gel, Griton XT Precast gel(BioRad, CA, USA) in order to identify the best transformants based onthe ability to produce large amount of glucoamylase. A selectedtransformant was identified on the original transformation plate and waspreserved as spores in a 20% glycerol stock and stored frozen (−80° C.).

Cultivation.

The selected transformant was inoculated in 100 ml of MLC media andcultivated at 30° C. for 2 days in 500 ml shake flasks on a rotaryshaker. 3 ml of the culture broth was inoculated to 100 ml of M410medium and cultivated at 30° C. for 3 days. The culture broth wascentrifugated and the supernatant was filtrated using 0.2 μm membranefilters.

Alpha-Cyclodextrin Affinity Gel.

Ten grams of Epoxy-activated Sepharose 6B (GE Healthcare, Chalfont St.Giles, U.K) powder was suspended in and washed with distilled water on asintered glass filter. The gel was suspended in coupling solution (100ml of 12.5 mg/ml alpha-cyclodextrin, 0.5 M NaOH) and incubated at roomtemperature for one day with gentle shaking. The gel was washed withdistilled water on a sintered glass filter, suspended in 100 ml of 1 Methanolamine, pH 10, and incubated at 50° C. for 4 hours for blocking.The gel was then washed several times using 50 mM Tris-HCl, pH 8 and 50mM NaOAc, pH 4.0 alternatively. The gel was finally packed in a 35-40 mlcolumn using equilibration buffer (50 mM NaOAc, 150 mM NaCl, pH 4.5).

Purification of Glucoamylase from Culture Broth.

Culture broth from fermentation of A. niger MBin118 harboring theglucoamylase gene was filtrated through a 0.22 μm PES filter, andapplied on a alpha-cyclodextrin affinity gel column previouslyequilibrated in 50 mM NaOAc, 150 mM NaCl, pH 4.5 buffer. Unboundmaterial was washed off the column with equilibration buffer and theglucoamylase was eluted using the same buffer containing 10 mMbeta-cyclodextrin over 3 column volumes.

The glucoamylase activity of the eluent was checked to see, if theglucoamylase had bound to the alpha-cyclodextrin affinity gel. Thepurified glucoamylase sample was then dialyzed against 20 mM NaOAc, pH5.0. The purity was finally checked by SDS-PAGE, and only a single bandwas found.

Example 8 Construction and Expression of a Site-Directed Variant ofPenicillium oxalicum Glucoamylase

Two PCR reactions were performed with plasmid XYZ1471-4, described inExample 7, using primers K79V F and K79VR shown below, which weredesigned to substitute lysine K at position 79 from the mature sequenceto valine (V) and primers F-NP003940 and R-NP003940 shown below, whichwere designed based on the known sequence and added tags for directcloning by IN-FUSION™ strategy.

Primer K79V F 18mer (SEQ ID NO: 18) GCAGTCTTTCCAATTGACPrimer K79V R 18mer (SEQ ID NO: 19) AATTGGAAAGACTGCCCGPrimer F-NP003940: (SEQ ID NO: 20) 5′ACACAACTGGGGATCCACCATGCGTCTCACTCTATTATC Primer R-NP003940:(SEQ ID NO: 21) 5′ AGATCTCGAGAAGCTTAAAACTGCCACACGTCGTTGG

The PCR was performed using a PTC-200 DNA Engine under the conditionsdescribed below.

PCR reaction system: Conditions: 48.5 micro L H2O 1 94° C. 2 min 2 beadspuRe Taq Ready-To- 2 94° C. 30 sec Go PCR Beads (Amersham Biosciences) 355° C. 30 sec 0.5 micro L X 2100 pmole/micro L Primers 4 72° C. 90 sec(K79V F + Primer R-NP003940, K79V R + 2-4 25 cycles Primer F-NP003940) 572° C. 10 min 0.5 micro L Template DNA

DNA fragments were recovered from agarose gel by the Qiagen gelextraction Kit according to the manufacturer's instruction. Theresulting purified two fragments were cloned into an Aspergillusexpression vector linearized with BamHI and HindIII, using an IN-FUSION™Dry-Down PCR Cloning Kit (BD Biosciences, Palo Alto, Calif., USA)according to the manufacturer's instructions. The linearized vectorconstruction is as described in WO 2005/042735 A1.

The ligation mixture was used to transform E. coli DH5α cells (TOYOBO).Selected colonies were inoculated in 3 ml of LB medium supplemented with50 μg of ampicillin per ml and incubated at 37° C. at 225 rpm overnight.Plasmid DNA from the selected colonies was purified using Qiagen plasmidmini kit (Qiagen) according to the manufacturer's instructions. Thesequence of Penicillium oxalicum glucoamylase site-directed variant genesequence was verified before heterologous expression and one of theplasmids was selected for further expression, and was named pPoPE001.

Protoplasts of Aspergillus niger MBin118 were prepared as described inWO 95/02043. One hundred μl of protoplast suspension were mixed with 2.5μg of the pPoPE001 plasmid and 250 microliters of 60% PEG 4000(Applichem) (polyethylene glycol, molecular weight 4,000), 10 mM CaCl₂,and 10 mM Tris-HCl pH 7.5 were added and gently mixed. The mixture wasincubated at 37° C. for 30 minutes and the protoplasts were mixed with1% agarose L (Nippon Gene) in COVE sucrose (Cove, 1996, Biochim.Biophys. Acta 133:51-56) supplemented with 10 mM acetamide and 15 mMCsCl and added as a top layer on COVE sucrose plates supplemented with10 mM acetamide and 15 mM CsCl for transformants selection (4 ml topagarper plate). After incubation for 5 days at 37° C. spores of sixteentransformants were picked up and seed on 750 μl YP-2% Maltose medium in96 deepwell MT plates. After 5 days of stationary cultivation at 30° C.,10 μl of the culture-broth from each well was analyzed on a SDS-PAGE gelin order to identify the best transformants based on the ability toproduce large amount of the glucoamylase.

Example 9 Purification of Site-Directed Po AMG Variant PE001

The selected transformant of the variant and the strain expressing thewild type Penicillium oxalicum glucoamylase described in Example 6 wascultivated in 100 ml of YP-2% maltose medium and the culture wasfiltrated through a 0.22 μm PES filter, and applied on aalpha-cyclodextrin affinity gel column previously equilibrated in 50 mMNaOAc, 150 mM NaCl, pH 4.5 buffer. Unbound materials was washed off thecolumn with equilibration buffer and the glucoamylase was eluted usingthe same buffer containing 10 mM beta-cyclodextrin over 3 columnvolumes.

The glucoamylase activity of the eluent was checked to see, if theglucoamylase had bound to the alpha-cyclodextrin affinity gel. Thepurified glucoamylase samples were then dialyzed against 20 mM NaOAc, pH5.0.

Example 10 Characterization of PE001 Protease Stability

40 μl enzyme solutions (1 mg/ml) in 50 mM sodium acetate buffer, pH 4.5,were mixed with 1/10 volume of 1 mg/ml protease solutions such asaspergillopepsin I described in Biochem J. 1975 April; 147(1):45-53, orthe commercially available product from Sigma and aorsin described inBiochemical journal [0264-6021] Ichishima yr: 2003 vol: 371 iss: Pt 2pg: 541 and incubated at 4 or 32° C. overnight. As a control experiment,H₂O was added to the sample instead of proteases. The samples wereloaded on SDS-PAGE to see if the glucoamylases are cleaved by proteases.

In SDS-PAGE, PE001 only showed one band corresponding to the intactmolecule, while the wild type glucoamylase was degraded by proteases andshowed a band at lower molecular size at 60 kCa.

TABLE 11 The result of SDS-PAGE after protease treatment Wild typeglucoamylase PE001 Protease aspergillopepsin I aorsin aspergillopepsin Iaorsin control Incubation 4 32 4 32 4 32 4 32 4 temperature (° C.)intact 100% 90% 40% 10% 100% 100% 100% 100% 100% glucoamylase (ca. 70kDa) cleaved N.D. 10% 60% 90% N.D. N.D. N.D N.D. N.D. glucoamylase (ca.60 kDa) N.D.: not detected.

Example 11 Less Cleavage During Cultivation

Aspergillus transformant of the variant and the wild type Penicilliumoxalicum glucoamylase were cultivated in 6-well MT plates containing 4×diluted YP-2% maltose medium supplemented with 10 mM sodium acetatebuffer, pH4.5, at 32° C. for 1 week.

The culture supernatants were loaded on SDS-PAGE.

TABLE 12 The result of SDS-PAGE of the culture supernatants Wild typeglucoamylase PE001 intact glucoamylase(ca. 70 kDa) 90% 100% cleavedglucoamylase (ca. 60 kDa) 10% N.D. N.D.: not detected.

The wild type glucoamylase was cleaved by host proteases duringfermentation, while the variant yielded only intact molecule.

Example 12 Glucoamylase Activity of Variant Compared to Parent

The glucoamylase activity measures as AGU as described above was checkedfor the purified enzymes of the wild type Penicillium oxalicum and thevariant glucoamylase.

The Glucoamylase Unit (AGU) was defined as the amount of enzyme, whichhydrolyzes 1 micromole maltose per minute under the standard conditions(37° C., pH 4.3, substrate: maltose 100 mM, buffer: acetate 0.1 M,reaction time 6 minutes).

TABLE 13 Relative specific activity AGU/mg Penicillium oxalicum wt 100%Penicillium oxalicum PE001 (SEQ ID NO: 14 + 102% K79V substitution)

Example 13 Purification of Glucoamylase Variants Having IncreasedThermostability

The variants showing increased thermostability may be constructed andexpressed similar to the procedure described in Example 8. All variantswere derived from the PE001. After expression in YPM medium, variantscomprising the T65A or Q327F substitution was micropurified as follows:

Mycelium was removed by filtration through a 0.22 μm filter. 50 μlcolumn material (alpha-cyclodextrin coupled to Mini-Leakdivinylsulfone-activated agarose medium according to manufacturer'srecommendations) was added to the wells of a filter plate (Whatman,Unifilter 800 μl, 25-30 μm MBPP). The column material was equilibratedwith binding buffer (200 mM sodium acetate pH 4.5) by two times additionof 200 μl buffer, vigorous shaking for 10 min (Heidolph, Titramax 101,1000 rpm) and removal of buffer by vacuum (Whatman, UniVac 3).Subsequently, 400 μl culture supernatant and 100 μl binding buffer wasadded and the plate incubated 30 min with vigorous shaking. Unboundmaterial was removed by vacuum and the binding step was repeated.Normally 4 wells were used per variant. Three washing steps were thenperformed with 200 μl buffer of decreasing ionic strength added (50/10/5mM sodium acetate, pH 4.5), shaking for 15 min and removal of buffer byvacuum. Elution of the bound AMG was achieved by two times addition of100 μl elution buffer (250 mM sodium acetate, 0.1% alpha-cyclodextrin,pH 6.0), shaking for 15 min and collection of eluted material in amicrotiter plate by vacuum. Pooled eluates were concentrated and bufferchanged to 50 mM sodium acetate pH 4.5 using centrifugal filter unitswith 10 kDa cut-off (Millipore Microcon Ultracel YM-10). Micropurifiedsamples were stored at −18° C. until testing of thermostability.

Example 14 Protein Thermal Unfolding Analysis (TSA, Thermal Shift Assay)

Protein thermal unfolding of the T65A and Q327F variants, was monitoredusing Sypro Orange (In-vitrogen, S-6650) and was performed using areal-time PCR instrument (Applied Biosystems; Step-One-Plus).

In a 96-well plate, 25 microliter micropurified sample in 50 mM AcetatepH4.5 at approx. 100 microgram/ml was mixed (5:1) with Sypro Orange(resulting conc.=5×; stock solution from supplier=5000×). The plate wassealed with an optical PCR seal. The PCR instrument was set at ascan-rate of 76° C. pr. hr, starting at 25° C. and finishing at 96° C.

Protein thermal unfolding of the E501V+Y504T variant, was monitoredusing Sypro Orange (In-vitrogen, S-6650) and was performed using areal-time PCR instrument (Applied Biosystems; Step-One-Plus).

In a 96-well plate, 15 microliter purified sample in 50 mM Acetate pH4.5at approx. 50 microgram/ml was mixed (1:1) with Sypro Orange (resultingconc.=5×; stock solution from supplier=5000×) with or without 200 ppmAcarbose (Sigma A8980). The plate was sealed with an optical PCR seal.The PCR instrument was set at a scan-rate of 76 degrees C. pr. hr,starting at 25° C. and finishing at 96° C.

Fluorescence was monitored every 20 seconds using in-built LED bluelight for excitation and ROX-filter (610 nm, emission).

Tm-values were calculated as the maximum value of the first derivative(dF/dK) (ref.: Gregory et al; J Biomol Screen 2009 14: 700.)

TABLE 14a Sample Tm (Deg. Celsius) +/− 0.4 PO-AMG (PE001) 80.3 VariantQ327F 82.3 Variant T65A 81.9

TABLE 14b Sample Tm (Deg. Celsius) +/− 0.4 Acarbose: − + PO-AMG (PE001)79.5 86.9 Variant E501V Y504T 79.5 95.2

Example 15 Thermostability Analysis by Differential Scanning Calorimetry(DSC)

Additional site specific variants having substitutions and for deletionsat specific positions were constructed basically as described in Example8 and purified as described in Example 11.

The thermostability of the purified Po-AMG PE001 derived variants weredetermined at pH 4.0 or 4.8 (50 mM Sodium Acetate) by DifferentialScanning calorimetry (DSC) using a VP-Capillary Differential Scanningcalorimeter (MicroCal Inc., Piscataway, N.J., USA). The thermaldenaturation temperature, Td (° C.), was taken as the top of thedenaturation peak (major endothermic peak) in thermograms (Cp vs. T)obtained after heating enzyme solutions in selected buffers (50 mMSodium Acetate, pH 4.0 or 4.8) at a constant programmed heating rate of200 K/hr.

Sample- and reference-solutions (approximately 0.3 ml) were loaded intothe calorimeter (reference: buffer without enzyme) from storageconditions at 10° C. and thermally pre-equilibrated for 10 minutes at20° C. prior to DSC scan from 20° C. to 110° C. Denaturationtemperatures were determined with an accuracy of approximately +/−1° C.

The isolated variants and the DSC data are disclosed in Table 15 below.

TABLE 15 Po- DSC Td DSC Td AMG (° C.) @ (° C.) @ name Mutations pH 4.0pH 4.8 PE001 82.1 83.4 (SEQ ID NO: 14 + K79V) GA167 E501V Y504T 82.1GA481 T65A K161S 84.1 86.0 GA487 T65A Q405T 83.2 GA490 T65A Q327W 87.3GA491 T65A Q327F 87.7 GA492 T65A Q327Y 87.3 GA493 P11F T65A Q327F 87.888.5 GA497 R1K D3W K5Q G7V N8S T10K P11S 87.8 88.0 T65A Q327F GA498 P2NP4S P11F T65A Q327F 88.3 88.4 GA003 P11F D26C K33C T65A Q327F 83.3 84.0GA009 P2N P4S P11F T65A Q327W E501V 88.8 Y504T GA002 R1E D3N P4G G6R G7AN8A T10D 87.5 88.2 P11D T65A Q327F GA005 P11F T65A Q327W 87.4 88.0 GA008P2N P4S P11F T65A Q327F E501V 89.4 90.2 Y504T GA010 P11F T65A Q327WE501V Y504T 89.7 GA507 T65A Q327F E501V Y504T 89.3 GA513 T65A S105PQ327W 87.0 GA514 T65A S105P Q327F 87.4 GA515 T65A Q327W S364P 87.8 GA516T65A Q327F S364P 88.0 GA517 T65A S103N Q327F 88.9 GA022 P2N P4S P11FK34Y T65A Q327F 89.7 GA023 P2N P4S P11F T65A Q327F D445N 89.9 V447SGA032 P2N P4S P11F T65A I172V Q327F 88.7 GA049 P2N P4S P11F T65A Q327FN502* 88.4 GA055 P2N P4S P11F T65A Q327F N502T 88.0 P563S K571E GA057P2N P4S P11F R31S K33V T65A 89.5 Q327F N564D K571S GA058 P2N P4S P11FT65A Q327F S377T 88.6 GA064 P2N P4S P11F T65A V325T Q327W 88.0 GA068 P2NP4S P11F T65A Q327F D445N 90.2 V447S E501V Y504T GA069 P2N P4S P11F T65AI172V Q327F 90.2 E501V Y504T GA073 P2N P4S P11F T65A Q327F S377T 90.1E501V Y504T GA074 P2N P4S P11F D26N K34Y T65A 89.1 Q327F GA076 P2N P4SP11F T65A Q327F I375A 90.2 E501V Y504T GA079 P2N P4S P11F T65A K218AK221D 90.9 Q327F E501V Y504T GA085 P2N P4S P11F T65A S103N Q327F 91.3E501V Y504T GA086 P2N P4S T10D T65A Q327F E501V 90.4 Y504T GA088 P2N P4SF12Y T65A Q327F E501V 90.4 Y504T GA097 K5A P11F T65A Q327F E501V 90.0Y504T GA101 P2N P4S T10E E18N T65A Q327F 89.9 E501V Y504T GA102 P2N T10EE18N T65A Q327F E501V 89.8 Y504T GA084 P2N P4S P11F T65A Q327F E501V90.5 Y504T T568N GA108 P2N P4S P11F T65A Q327F E501V 88.6 Y504T K524TG526A GA126 P2N P4S P11F K34Y T65A Q327F 91.8 D445N V447S E501V Y504TGA129 P2N P4S P11F R31S K33V T65A 91.7 Q327F D445N V447S E501V Y504TGA087 P2N P4S P11F D26N K34Y T65A 89.8 Q327F E501V Y504T GA091 P2N P4SP11F T65A F80* Q327F 89.9 E501V Y504T GA100 P2N P4S P11F T65A K112SQ327F 89.8 E501V Y504T GA107 P2N P4S P11F T65A Q327F E501V 90.3 Y504TT516P K524T G526A GA110 P2N P4S P11F T65A Q327F E501V 90.6 N502T Y504*

Example 16 Thermostability Analysis by Thermo-Stress Test and pNPG Assay

Starting from one of the identified substitution variants from Example15, identified as GA008, additional variants were tested by athermo-stress assay in which the supernatant from growth cultures wereassayed for glucoamylase (AMG) activity after a heat shock at 83° C. for5 min.

After the heat-shock the residual activity of the variant was measuredas well as in a non-stressed sample.

Description of Po-AMG pNPG Activity Assay:

The Penicillium oxalicum glucoamylase pNPG activity assay is aspectrometric endpoint assay where the samples are split in two andmeasured thermo-stressed and non-thermo-stressed. The data output istherefore a measurement of residual activity in the stressed samples.

Growth:

A sterile micro titer plate (MTP) was added 200 μL rich growth media (FTX-14 without Dowfax) to each well. The strains of interest wereinoculated in triplicates directly from frozen stocks to the MTP.Benchmark was inoculated in 20 wells. Non-inoculated wells with mediawere used as assay blanks. The MTP was placed in a plastic boxcontaining wet tissue to prevent evaporation from the wells duringincubation. The plastic box was placed at 34° C. for 4 days.

Assay:

50 μL supernatant was transferred to 50 μL 0.5 M NaAc pH 4.8 to obtaincorrect sample pH.

50 μL dilution was transferred to a PCR plate and thermo-stressed at 83°C. for 5 minutes in a PCR machine. The remaining half of the dilutionwas kept at RT.

20 μL of both stressed and unstressed samples was transferred to astandard MTP. 20 μL pNPG-substrate was added to start the reaction. Theplate was incubated at RT for 1 hour.

The reaction was stopped and the colour developed by adding 50 μL 0.5MNa₂CO₃. The yellow colour was measured on a plate reader (MolecularDevices) at 405 nm.

Buffers: 0.5 M NaAc pH 4.8 0.25 M NaAc pH 4.8

Substrate, 6 mM pNPG:15 mg 4-nitrophenyl D-glucopyranoside in 10 mL 0.25 NaAc pH 4.8STOP/developing solution:

0.5 M Na₂CO₃ Data Treatment:

In Excel the raw Abs405 data from both stressed and unstressed sampleswere blank subtracted with their respective blanks. The residualactivity (% res.act.=(Abs_(unstressed)−(Abs_(unstressed)−Abs_(stressed)))/Abs_(unstressed)*100%)was calculated and plotted relative to benchmark, Po-amg0008.

TABLE 16 Po-AMG name Mutations % residual activity GA008 P2N P4S P11FT65A Q327F 100 E501V Y504T GA085 P2N P4S P11F T65A S103N 127 Q327F E501VY504T GA097 K5A P11F T65A Q327F 106 E501V Y504T GA107 P2N P4S P11F T65AQ327F 109 E501V Y504T T516P K524T G526A GA130 P2N P4S P11F T65A V79A 111Q327F E501V Y504T GA131 P2N P4S P11F T65A V79G 112 Q327F E501V Y504TGA132 P2N P4S P11F T65A V79I 101 Q327F E501V Y504T GA133 P2N P4S P11FT65A V79L 102 Q327F E501V Y504T GA134 P2N P4S P11F T65A V79S 104 Q327FE501V Y504T GA150 P2N P4S P11F T65A L72V 101 Q327F E501V Y504T GA155S255N Q327F E501V Y504T 105

TABLE 17 Po-AMG name Mutations % residual activity GA008 P2N P4S P11FT65A Q327F 100 E501V Y504T GA179 P2N P4S P11F T65A E74N 108 V79K Q327FE501V Y504T GA180 P2N P4S P11F T65A G220N 108 Q327F E501V Y504T GA181P2N P4S P11F T65A Y245N 102 Q327F E501V Y504T GA184 P2N P4S P11F T65AQ253N 110 Q327F E501V Y504T GA185 P2N P4S P11F T65A D279N 108 Q327FE501V Y504T GA186 P2N P4S P11F T65A Q327F 108 S359N E501V Y504T GA187P2N P4S P11F T65A Q327F 102 D370N E501V Y504T GA192 P2N P4S P11F T65AQ327F 102 V460S E501V Y504T GA193 P2N P4S P11F T65A Q327F 102 V460TP468T E501V Y504T GA195 P2N P4S P11F T65A Q327F 103 T463N E501V Y504TGA196 P2N P4S P11F T65A Q327F 106 S465N E501V Y504T GA198 P2N P4S P11FT65A Q327F 106 T477N E501V Y504T

Example 17 Test for Glucoamylase Activity of Thermo-Stable VariantsAccording to the Invention

All of the above described variants disclosed in tables 15, 16, and 17have been verified for Glucoamylase activity on culture supernatantsusing the pNPG assay described in Example 16.

Example 18 Ethanol Production Using Alpha-Amylase A (AAA), Protease 196,and Glucoamylase 493 (GA493) for Liquefaction and Glucoamylase BL3 (BL3)and Cellulolytic Composition A (CCA) for Fermentation Liquefaction(Labomat)

Each liquefaction received ground corn (86.3% DS), backset (7.2% DS),and tap water targeting a total weight of 150 g at 32.50% Dry Solids(DS). Backset was blended at 30% w/w of total slurry weight. Initialslurry pH was 5.2 and was therefore not adjusted before liquefaction.All enzymes were added according to the table below.

Glucoamylase Alpha-Amylase A Protease 196 GA493 Mash #1 0.02% w/w cornas is none none Mash #2 0.02% w/w corn as is 0.001 JTPU/g DS 6 mcg/g DS

Liquefaction took place in a Labomat using the following conditions: 5°C./min. Ramp, 17 minute Ramp, 103 minute hold time at 85° C., 40 rpm forthe entire run, 200 mL stainless steel canisters. After liquefaction,all canisters were cooled in an ice bath and prepared for fermentationbased on the protocol listed below under SSF.

Simultaneous Saccharification and Fermentation (SSF)

Two mashes above were adjusted to pH 5.0 with 50% w/w Sodium Hydroxideor 40% v/v sulfuric acid. Penicillin was applied to each mash to a totalconcentration of 3 ppm, and urea was added to each mash as nitrogensource to a final concentration of 1000 ppm. The tubes were preparedwith mash by aliquoting approximately 4.5 g of mash per 15 mLpre-drilled test tubes to allow CO₂ release. Novozymes glucoamylaseSpirizyme Excel and cellulase Celluclast were dosed into the tubesaccording to the following table:

AMG Cellulolytic Cellulase Treat- Gluco- Dosage Composition Dosage ment# Mash amylase AGU/g DS (CC) mg EP/g DS 1 Mash #1 BL3 0.60 — — 2 Mash #1BL3 0.60 A 0.10 3 Mash #2 BL3 0.60 — — 4 Mash #2 BL3 0.60 A 0.10

Distilled water was added to each tube in the appropriate volume to keepthe solids at the same concentration in all tubes. All treatments wereconducted in five replicates. After enzyme dosage, each tube received100 μL of rehydrated yeast. Rehydrated yeast was prepared by mixing 5.5g of Fermentis RED STAR into 100 mL of tap water and incubating at 32°C. for about 30 minutes. All the tubes were vortexed, and then incubatedin 32° C. water bath for 52 hours in the SSF process.

Fermentation sampling took place after 52 hours of fermentation. Eachsample was deactivated with 50 μL of 40% v/v H₂SO₄, vortexing,centrifuging at 3000 rpm for 10 minutes, and filtering through a 0.45 μmWhatman PP filter. All samples were analyzed by HPLC.

Results:

Treatment Ethanol (g/L) Std Dev. CV AAA + BL3 124.48 0.0257 0.21% AAA +BL3 + CCA 125.21 0.0358 0.29% AAA + Protease196 + GA493 + 125.16 0.03170.25% BL3 AAA + Protease196 + GA493 + 125.43 0.0495 0.39% BL3 + CCA

With Cellulolytic Composition A (CCA) addition into the SSF process,there was a 0.73 g/L ethanol yield increase from the corn mash liquefiedby Alpha-Amylase A (AAA). When adding Protease196 and Glucoamylase 493(GA493) together with Alpha-amylase A into the liquefaction, and addingCellulolytic Composition A (CCA) into SSF, the total ethanol yield wasincreased by 1 g/L.

Example 19 Ethanol Production Using Alpha-Amylase A or Alpha-AmylaseAA369, Protease Pfu2 and Glucoamylase 498 (GA498) for Liquefaction, andGlucoamylase BL4 with Cellulolytic Composition A or B (CCA or CCB) forFermentation Liquefaction (Labomat)

Each liquefaction received ground corn (86.3% DS), backset (7.2% DS),and tap water targeting a total weight of 375 g at 32.50% Dry Solids(DS). Backset was blended at 30% w/w of total slurry weight. Initialslurry pH was adjusted before liquefaction. All enzymes were addedaccording to the table below.

Amylase and Protease and Glucoamylase and Dose dose dose Mash #1 LSCDSpH 5.8 0.024% w/w corn as is none none Mash #2 AA369 PFU2 GA498 pH 5.22.14 μg/g DS 0.0385 μg/g DS 4.5 μg/g DS

Liquefaction took place in a Labomat using the following conditions: In200 mL stainless steel canisters increase temperature by 5° C./min up to80° C.; hold 2 min, then 2° C./min up to 85° C.; hold at 85° C. for 103min. After liquefaction, all mashes were stored frozen until they wereprepared for fermentation based on the protocol listed below under SSF.

Simultaneous Saccharification and Fermentation (SSF)

Each mash above was adjusted to pH 5.0 with 50% w/w Sodium Hydroxide or40% v/v sulfuric acid. Penicillin was applied to each mash to a totalconcentration of 3 ppm, and urea was added to each mash as nitrogensource to a final concentration of 800 ppm. Solids content of bothmashes was adjusted to 30% by addition of water. The tubes were preparedwith mash by aliquoting approximately 4.5 g of mash per 15 mLpre-drilled test tubes to allow CO₂ release. Glucoamylase BL4 andCellulolytic Composition CCA or CCB were dosed into the tubes accordingto the following table:

AMG Cellulolytic Cellulase Treat- Gluco- Dosage Composition Dosage ment# Mash amylase AGU/g DS (CC) mg EP/g DS 1 Mash #1 BL4 0.60 none 0 2 Mash#1 BL4 0.60 CCB 0.05 3 Mash #1 BL4 0.60 CCB 0.15 4 Mash #1 BL4 0.60 CCB0.3 5 Mash #1 BL4 0.60 CCA 0.05 6 Mash #1 BL4 0.60 CCA 0.15 7 Mash #1BL4 0.60 CCA 0.3 8 Mash #2 BL4 0.60 none 0 9 Mash #2 BL4 0.60 CCB 0.0510 Mash #2 BL4 0.60 CCB 0.15 11 Mash #2 BL4 0.60 CCB 0.3 12 Mash #2 BL40.60 CCA 0.05 13 Mash #2 BL4 0.60 CCA 0.15 14 Mash #2 BL4 0.60 CCA 0.3

Distilled water was added to each tube in the appropriate volume to keepthe solids at the same concentration in all tubes. All treatments wereconducted in five replicates. After enzyme dosage, each tube received100 μL of rehydrated yeast. Rehydrated yeast was prepared by mixing 5.5g of Fermentis RED STAR into 100 mL of tap water and incubated at 32° C.for about 30 minutes. All the tubes were vortexed, and then incubated in32° C. water bath for 51 hours in the SSF process.

Fermentation sampling took place after 51 hours of fermentation. Eachsample was deactivated with 50 μL of 40% v/v H₂SO₄, vortexing,centrifuging at 3000 rpm for 10 minutes, and filtering through a 0.45 μmWhatman PP filter. All samples were analyzed by HPLC.

Results:

Treatment Ethanol (g/L) Std Dev. CV AAA + BL4 114.99 0.67 0.58% AAA +BL4 + CCB 0.05 116.08 0.87 0.75% AAA + BL4 + CCB 0.15 117.17 0.86 0.73%AAA + BL4 + CCB 0.3 117.61 0.92 0.78% AAA + BL4 + CCA 0.05 115.53 0.760.65% AAA + BL4 + CCA 0.15 115.46 0.92 0.79% AAA + BL4 + CCA 0.3 115.840.79 0.68% AA369 + GA498 + Pfu2 + BL4 115.51 0.68 0.59% AA369 + GA498 +Pfu2 + BL4 + 116.70 0.64 0.55% CCB 0.05 AA369 + GA498 + Pfu2 + BL4 +117.31 0.86 0.73% CCB 0.15 AA369 + GA498 + Pfu2 + BL4 + 118.74 0.720.61% CCB 0.3 AA369 + GA498 + Pfu2 + BL4 + 116.90 0.36 0.31% CCA 0.05AA369 + GA498 + Pfu2 + BL4 + 117.38 0.88 0.75% CCA 0.15 AA369 + GA498 +Pfu2 + BL4 + 116.99 0.27 0.23% CCA 0.3

With Cellulolytic Composition A (CCA) addition into the SSF process,there was an ethanol yield increase of up to 0.74% compared to the cornmash liquefied by Alpha-Amylase A (AAA) with no added CellulolyticComposition in fermentation. With Cellulolytic Composition B (CCB) inthe same mash, there was an ethanol yield increase of up to 2.28%.

When adding Protease Pfu2 and Glucoamylase 498 (GA498) together withAlpha-amylase 369 into the liquefaction, and adding CellulolyticComposition A (CCA) into SSF, the total ethanol yield was increased byup to 1.62% compared to the same mash with no added CellulolyticComposition. With Cellulolytic Composition B (CCB) in the same mash,there was an ethanol yield increase of up to 2.80%.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The present invention is further described in the following numberedparagraphs:1. A process for producing fermentation products from starch-containingmaterial comprising the steps of:i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.; and    -   optionally a carbohydrate-source generating enzyme;        ii) saccharifying using a carbohydrate-source generating enzyme;        iii) fermenting using a fermenting organism;        wherein a cellulolytic composition is present or added during        fermentation or simultaneous saccharification and fermentation.        2. The process of paragraph 1, further comprises, prior to the        liquefaction step i), the steps of:

a) reducing the particle size of the starch-containing material,preferably by dry milling;

b) forming a slurry comprising the starch-containing material and water.

3. The process of any of paragraphs 1-2, wherein at least 50%,preferably at least 70%, more preferably at least 80%, especially atleast 90% of the starch-containing material fit through a sieve with #6screen.4. The process of any of paragraphs 1-3, wherein the pH duringliquefaction is from 4.5-5.0, such as between 4.5-4.8.5. The process of any of paragraphs 1-3, wherein the pH duringliquefaction is between above 5.0-6.5, such as above 5.0-6.0, such asabove 5.0-5.5, such as between 5.2-6.2, such as around 5.2, such asaround 5.4, such as around 5.6, such as around 5.8.6. The process of any of paragraphs 1-5, wherein the temperature duringliquefaction is in the range from 70-100° C., such as between 75-95° C.,such as between 75-90° C., preferably between 80-90° C., such as 82-88°C., such as around 85° C.7. The process of any of paragraphs 1-6, wherein a jet-cooking step iscarried out after liquefaction in step i).8. The process of paragraph 7, wherein the jet-cooking is carried out ata temperature between 110-145° C., preferably 120-140° C., such as125-135° C., preferably around 130° C. for about 1-15 minutes,preferably for about 3-10 minutes, especially around about 5 minutes.9. The process of any of paragraphs 1-8, wherein saccharification andfermentation is carried out sequentially or simultaneously.10. The process of any of paragraphs 1-9, wherein saccharification iscarried out at a temperature from 20-75° C., preferably from 40-70° C.,such as around 60° C., and at a pH between 4 and 5.11. The process of any of paragraphs 1-10, wherein fermentation orsimultaneous saccharification and fermentation (SSF) is carried outcarried out at a temperature from 25° C. to 40° C., such as from 28° C.to 35° C., such as from 30° C. to 34° C., preferably around about 32° C.In an embodiment fermentation is ongoing for 6 to 120 hours, inparticular 24 to 96 hours.12. The process of any of paragraphs 1-11, wherein the fermentationproduct is recovered after fermentation, such as by distillation.13. The process of any of paragraphs 1-12, wherein the fermentationproduct is an alcohol, preferably ethanol, especially fuel ethanol,potable ethanol and/or industrial ethanol.14. The process of any of paragraphs 1-13, wherein the starch-containingstarting material is whole grains.15. The process of any of paragraphs 1-14, wherein the starch-containingmaterial is derived from corn, wheat, barley, rye, milo, sago, cassava,manioc, tapioca, sorghum, rice or potatoes.16. The process of any of paragraphs 1-15, wherein the fermentingorganism is yeast, preferably a strain of Saccharomyces, especially astrain of Saccharomyces cerevisiae.17. The process of any of paragraphs 1-16, wherein the alpha-amylase isa bacterial or fungal alpha-amylase.18. The process of any of paragraphs 1-17, wherein the alpha-amylase isfrom the genus Bacillus, such as a strain of Bacillusstearothermophilus, in particular a variant of a Bacillusstearothermophilus alpha-amylase, such as the one shown in SEQ ID NO: 3in WO 99/019467 or SEQ ID NO: 1 herein.19. The process of paragraph 18, wherein the Bacillus stearothermophilusalpha-amylase or variant thereof is truncated, preferably to have around491 amino acids.20. The process of any of paragraphs 18 or 19, wherein the Bacillusstearothermophilus alpha-amylase has a double deletion of positionsI181+G182 and optionally a N193F substitution, or deletion of R179+G180(using SEQ ID NO: 1 for numbering).21. The process of any of paragraphs 18-20 wherein the Bacillusstearothermophilus alpha-amylase has a substitution in position S242,preferably S242Q substitution.22. The process of any of paragraphs 18-21, wherein the Bacillusstearothermophilus alpha-amylase has a substitution in position E188,preferably E188P substitution.23. The process of any of paragraphs 1-22, wherein the alpha-amylase hasa T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of at least 10, such as atleast 15, such as at least 20, such as at least 25, such as at least 30,such as at least 40, such as at least 50, such as at least 60, such asbetween 10-70, such as between 15-70, such as between 20-70, such asbetween 25-70, such as between 30-70, such as between 40-70, such asbetween 50-70, such as between 60-70.24. The process of any of paragraphs 1-23, wherein the alpha-amylase isselected from the group of Bacillus stearothermophilus alpha-amylasevariants with the following mutations in addition to I181*+G182* andoptionally N193F:

-   -   V59A+Q89R+G112D+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+D269E+D281N;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+I270L;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+H274K;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+Y276F;    -   V59A+E129V+R157Y+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;    -   59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+H274K;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+D281N;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+G416V;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   A91L+M96I+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F+L427M;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+N376*+I377*;    -   E129V+K177L+R179E+K220P+N224L+Q254S;    -   E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   E129V+K177L+R179E+S242Q;    -   E129V+K177L+R179V+K220P+N224L+S242Q+Q254S;    -   K220P+N224L+S242Q+Q254S;    -   M284V;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V.    -   V59A+E129V+K177L+R179E+Q254S+M284V;        25. The process of any of paragraphs 1-24, wherein the        alpha-amylase is selected from the following group of Bacillus        stearothermophilus alpha-amylase variants:    -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   I181*+G182*+N193F+V59A+E129V+K177L+R179E+Q254S+M284V and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 for numbering).        26. The process of any of paragraphs 1-25, wherein the protease        with a thermostability value of more than 25% determined as        Relative Activity at 80° C./70° C.        27. The process of any of paragraphs 1-26, wherein the protease        has a thermostability of more than 30%, more than 40%, more than        50%, more than 60%, more than 70%, more than 80%, more than 90%,        more than 100%, such as more than 105%, such as more than 110%,        such as more than 115%, such as more than 120% determined as        Relative Activity at 80° C./70° C.        28. The process of any of paragraphs 1-27, wherein the protease        has a thermostability of between 20 and 50%, such as between 20        and 40%, such as 20 and 30% determined as Relative Activity at        80° C./70° C.        29. The process of any of paragraphs 1-28, wherein the protease        has a thermostability between 50 and 115%, such as between 50        and 70%, such as between 50 and 60%, such as between 100 and        120%, such as between 105 and 115% determined as Relative        Activity at 80° C./70° C.        30. The process of any of paragraphs 1-29, wherein the protease        has a thermostability of more than 10%, such as more than 12%,        more than 14%, more than 16%, more than 18%, more than 20%, more        than 30%, more than 40%, more that 50%, more than 60%, more than        70%, more than 80%, more than 90%, more than 100%, more than        110% determined as Relative Activity at 85° C./70° C.        31. The process of any of paragraphs 1-30, wherein the protease        has thermostability of between 10 and 50%, such as between 10        and 30%, such as between 10 and 25% determined as Relative        Activity at 85° C./70° C.        32. The process of any of paragraphs 1-31, wherein the protease        has a themostability above 60%, such as above 90%, such as above        100%, such as above 110% at 85° C. as determined using the        Zein-BCA assay.        33. The process of any of paragraphs 1-32, wherein the protease        has a themostability between 60-120, such as between 70-120%,        such as between 80-120%, such as between 90-120%, such as        between 100-120%, such as 110-120% at 85° C. as determined using        the Zein-BCA assay.        34. The process of any of paragraphs 1-33, wherein the protease        is of fungal origin.        35. The process of any of paragraphs 1-34, wherein the protease        is a variant of the metallo protease derived from a strain of        the genus Thermoascus, preferably a strain of Thermoascus        aurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670.        36. The process of any of paragraphs 1-35, wherein the protease        is a variant of the metallo protease disclosed as the mature        part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the mature        part of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 3 herein        mutations selected from the group of:    -   S5*+D79L+S87P+A112P+D142L;    -   D79L+S87P+A112P+T124V+D142L;    -   S5*+N26R+D79L+S87P+A112P+D142L;    -   N26R+T46R+D79L+S87P+A112P+D142L;    -   T46R+D79L+S87P+T116V+D142L;    -   D79L+P81R+S87P+A112P+D142L;    -   A27K+D79L+S87P+A112P+T124V+D142L;    -   D79L+Y82F+S87P+A112P+T124V+D142L;    -   D79L+Y82F+S87P+A112P+T124V+D142L;    -   D79L+S87P+A112P+T124V+A126V+D142L;    -   D79L+S87P+A112P+D142L;    -   D79L+Y82F+S87P+A112P+D142L;    -   S38T+D79L+S87P+A112P+A126V+D142L;    -   D79L+Y82F+S87P+A112P+A126V+D142L;    -   A27K+D79L+S87P+A112P+A126V+D142L;    -   D79L+S87P+N98C+A112P+G135C+D142L;    -   D79L+S87P+A112P+D142L+T141C+M161C;    -   S36P+D79L+S87P+A112P+D142L;    -   A37P+D79L+S87P+A112P+D142L;    -   S49P+D79L+S87P+A112P+D142L;    -   S50P+D79L+S87P+A112P+D142L;    -   D79L+S87P+D104P+A112P+D142L;    -   D79L+Y82F+S87G+A112P+D142L;    -   S70V+D79L+Y82F+S87G+Y97W+A112P+D142L;    -   D79L+Y82F+S87G+Y97W+D104P+A112P+D142L;    -   S70V+D79L+Y82F+S87G+A112P+D142L;    -   D79L+Y82F+S87G+D104P+A112P+D142L;    -   D79L+Y82F+S87G+A112P+A126V+D142L;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L;    -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;    -   A27K+Y82F+S87G+D104P+A112P+A126V+D142L;    -   A27K+D79L+Y82F+D104P+A112P+A126V+D142L;    -   A27K+Y82F+D104P+A112P+A126V+D142L;    -   A27K+D79L+S87P+A112P+D142L; and    -   D79L+S87P+D142L.        37. The process of any of paragraphs 1-36, wherein the protease        is a variant of the metallo protease disclosed as the mature        part of SEQ ID NO. 2 disclosed in WO 2003/048353 or the mature        part of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 3 herein        with the following mutations:

D79L+S87P+A112P+D142L: D79L+S87P+D142L; orA27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

38. The process of any of paragraphs 1-37, wherein the protease varianthas at least 75% identity preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, more preferably at least 91%,more preferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, but lessthan 100% identity to the mature part of the polypeptide of SEQ ID NO: 2disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 in WO2010/008841 or SEQ ID NO: 3 herein.39. The process of any of paragraphs 1-38, wherein the protease variantof the Thermoascus aurantiacus protease shown in SEQ ID NO: 3 is one ofthe following:

-   -   D79L S87P D142L    -   D79L S87P A112P D142L    -   D79L Y82F S87P A112P D142L    -   S38T D79L S87P A112P A126V D142L    -   D79L Y82F S87P A112P A126V D142L    -   A27K D79L S87P A112P A126V D142L    -   S49P D79L S87P A112P D142L    -   S50P D79L S87P A112P D142L    -   D79L S87P D104P A112P D142L    -   D79L Y82F S87G A112P D142L    -   S70V D79L Y82F S87G Y97W A112P D142L    -   D79L Y82F S87G Y97W D104P A112P D142L    -   S70V D79L Y82F S87G A112P D142L    -   D79L Y82F S87G D104P A112P D142L    -   D79L Y82F S87G A112P A126V D142L    -   Y82F S87G S70V D79L D104P A112P D142L    -   Y82F S87G D79L D104P A112P A126V D142L    -   A27K D79L Y82F S87G D104P A112P A126V D142L        40. The process of any of paragraphs 1-39, wherein the protease        is of bacterial origin.        41. The process of any of paragraphs 1-40, wherein the protease        is derived from a strain of Pyrococcus, preferably a strain of        Pyrococcus furiosus.        42. The process of any of paragraphs 1-41, wherein the protease        is the one shown in SEQ ID NO: 1 in U.S. Pat. No. 6,358,726, SEQ        ID NO: 13 herein or SEQ ID NO: 29 herein.        43. The process of any of paragraphs 1-42, wherein the protease        is one having at least 80%, such as at least 85%, such as at        least 90%, such as at least 95%, such as at least 96%, such as        at least 97%, such as at least 98%, such as at least 99%        identity to in SEQ ID NO: 1 in U.S. Pat. No. 6,358,726 or SEQ ID        NO: 13 herein.        44. The process of any of paragraphs 1-43, wherein a        carbohydrate-source generating enzyme is present and/or added        during liquefaction step i), preferably a glucoamylase.        45. The process of any of paragraphs 1-44, wherein the        carbohydrate-source generating enzyme present and/or added        during liquefaction step i) is a glucoamylase having a heat        stability at 85° C., pH 5.3, of at least 20%, such as at least        30%, preferably at least 35%.        46. The process of any of paragraphs 44-45, wherein the        carbohydrate-source generating enzyme is a glucoamylase having a        relative activity pH optimum at pH 5.0 of at least 90%,        preferably at least 95%, preferably at least 97%.        47. The process of any of paragraphs 44-46, wherein the        carbohydrate-source generating enzyme is a glucoamylase having a        pH stability at pH 5.0 of at least at least 80%, at least 85%,        at least 90%.        48. The process of any of paragraphs 44-47, wherein the        carbohydrate-source generating enzyme present and/or added        during liquefaction step i) is a glucoamylase, preferably        derived from a strain of the genus Penicillium, especially a        strain of Penicillium oxalicum disclosed as SEQ ID NO: 2 in WO        2011/127802 or SEQ ID NOs: 9 or 14 herein.        49. The process of paragraph 44-48, wherein the glucoamylase has        at least 80%, more preferably at least 85%, more preferably at        least 90%, more preferably at least 91%, more preferably at        least 92%, even more preferably at least 93%, most preferably at        least 94%, and even most preferably at least 95%, such as even        at least 96%, at least 97%, at least 98%, at least 99% or 100%        identity to the mature polypeptide shown in SEQ ID NO: 2 in WO        2011/127802 or SEQ ID NOs: 9 or 14 herein.        50. The composition of any of paragraphs 44-49, wherein the        carbohydrate-source generating enzyme is a variant of the        glucoamylase derived from a strain of Penicillium oxalicum        disclosed as SEQ ID NO: 2 in WO 2011/127802 having a K79V        substitution (using the mature sequence shown in SEQ ID NO: 14        for numbering).        51. The process of any of paragraphs 44-50, further wherein a        glucoamylase is present and/or added during saccharification        and/or fermentation.        52. The process of any of paragraphs 1-51, wherein the        glucoamylase present and/or added during saccharification and/or        fermentation is of fungal origin, preferably from a stain of        Aspergillus, preferably A. niger, A. awamori, or A. oryzae; or a        strain of Trichoderma, preferably T. reesei; or a strain of        Talaromyces, preferably T. emersonii, or a strain of Pycnoporus,        or a strain of Gloeophyllum, such as a strain of Gloeophyllum        sepiarium or Gloeophyllum trabeum, such as one disclosed in WO        2011/068803 as any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16,        preferably SEQ ID NO: 2 in WO 2011/068803, or a strain of the        Nigrofomes.        53. The process of paragraph 52, wherein the glucoamylase        present and/or added during saccharification and/or fermentation        is a blend comprising Talaromyces emersonii glucoamylase        disclosed in WO 99/28448 as SEQ ID NO: 7 and Trametes cingulata        glucoamylase disclosed in WO 06/069289.        54. The process of paragraphs 52 or 53 wherein the glucoamylase        present and/or added during saccharification and/or fermentation        is a blend comprising Talaromyces emersonii glucoamylase        disclosed in WO 99/28448, Trametes cingulata glucoamylase        disclosed in WO 06/69289, and Rhizomucor pusillus alpha-amylase        with Aspergillus niger glucoamylase linker and SBD disclosed as        V039 in Table 5 in WO 2006/069290.        55. The process of any of paragraphs 52-54, wherein the        glucoamylase present and/or added during saccharification and/or        fermentation is a blend_comprising Talaromyces emersonii        glucoamylase disclosed in WO99/28448, Trametes cingulata        glucoamylase disclosed in WO 06/69289, and Rhizomucor pusillus        alpha-amylase with Aspergillus niger glucoamylase linker and SBD        disclosed as V039 in Table 5 in WO 2006/069290.        56. The process of paragraph 52, wherein the glucoamylase        present and/or added during saccharification and/or fermentation        is a blend comprising Gloeophyllum sepiarium glucoamylase shown        as SEQ ID NO: 2 in WO 2011/068803 and Rhizomucor pusillus with        an Aspergillus niger glucoamylase linker and starch-binding        domain (SBD), disclosed SEQ ID NO: 3 in WO 2013/006756 with the        following substitutions: G128D+D143N.        57. The process of any of paragraphs 1-56, further wherein a        pullulanase is present during liquefaction and/or        saccharification.        58. The process of any of paragraphs 1-57, comprising the steps        of:        i) liquefying the starch-containing material at a temperature        above the initial gelatinization temperature using:    -   an alpha-amylase derived from Bacillus stearothermophilus;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.,        preferably derived from Pyrococcus furiosus and/or Thermoascus        aurantiacus; and    -   optionally a Penicillium oxalicum glucoamylase;        ii) saccharifying using a glucoamylase enzyme;        iii) fermenting using a fermenting organism;        wherein a cellulolytic composition is present or added during        fermentation or simultaneous saccharification and fermentation.        59. A process of paragraphs 1-58, comprising the steps of:        i) liquefying the starch-containing material at a temperature        above the initial gelatinization temperature using:    -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   optionally a protease, preferably derived from Pyrococcus        furiosus and/or Thermoascus aurantiacus, having a        thermostability value of more than 20% determined as Relative        Activity at 80° C./70° C.;    -   optionally a Penicillium oxalicum glucoamylase;        ii) saccharifying using a glucoamylase enzyme;        iii) fermenting using a fermenting organism;        wherein a cellulolytic composition is present or added during        fermentation or simultaneous saccharification and fermentation.        60. A process of paragraphs 1-59, comprising the steps of:

i) liquefying the starch-containing material at a temperature between80-90° C.:

-   -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   optionally a protease, preferably derived from Pyrococcus        furiosus and/or Thermoascus aurantiacus, having a        thermostability value of more than 20% determined as Relative        Activity at 80° C./70° C.;    -   optionally a Penicillium oxalicum glucoamylase

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.61. A process of paragraphs 1-60, comprising the steps of:

i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182 and optional substitution N193F; and        optionally further one of the following set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S:    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   V59A+E129V+K177L+R179E+Q254S+M284V;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering).    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.,        preferably derived from Pyrococcus furiosus and/or Thermoascus        aurantiacus; and    -   optionally a Penicillium oxalicum glucoamylase shown in SEQ ID        NO: 14 having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering);

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.62. A process of paragraphs 1-61, comprising the steps of:

i) liquefying the starch-containing material at a temperature between80-90° C. using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182 and optional substitution N193F; and        optionally further one of the following set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   V59A+E129V+K177L+R179E+Q254S+M284V;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering),    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.,        preferably derived from Pyrococcus furiosus and/or Thermoascus        aurantiacus;    -   optionally a pullulanase    -   optionally a Penicillium oxalicum glucoamylase shown in SEQ ID        NO: 14 having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering);

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism;

wherein a cellulolytic composition is present and/or added duringfermentation or simultaneous saccharification and fermentation.63. The process of any of paragraphs 1-62, comprising the steps of:i) liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.,        preferably derived from Pyrococcus furiosus and/or Thermoascus        aurantiacus; and    -   optionally a pullulanase;    -   optionally a Penicillium oxalicum glucoamylase;        ii) saccharifying using a glucoamylase enzyme;        iii) fermenting using a fermenting organism wherein a        cellulolytic composition is present and/or added during        fermentation or simultaneous        saccharification and fermentation.        64. A process of paragraphs 1-63, comprising the steps of:    -   i) liquefying the starch-containing material at a temperature        above the initial gelatinization temperature using:        -   an alpha-amylase, preferably derived from Bacillus            stearothermophilus, having a T½ (min) at pH 4.5, 85° C.,            0.12 mM CaCl₂ of at least 10;        -   optionally a protease, preferably derived from Pyrococcus            furiosus and/or Thermoascus aurantiacus, having a            thermostability value of more than 20% determined as            Relative Activity at 80° C./70° C.;        -   optionally a pullulanase;        -   optionally a Penicillium oxalicum glucoamylase    -   ii) saccharifying using a glucoamylase enzyme;    -   iii) fermenting using a fermenting organism;    -   wherein a cellulolytic composition is present and/or added        during fermentation or simultaneous saccharification and        fermentation.        65. A process of paragraphs 1-64, comprising the steps of:    -   i) liquefying the starch-containing material at a temperature        between 80-90° C.:        -   an alpha-amylase, preferably derived from Bacillus            stearothermophilus, having a T½ (min) at pH 4.5, 85° C.,            0.12 mM CaCl₂ of at least 10;        -   optionally a optionally a protease, preferably derived from            Pyrococcus furiosus or Thermoascus aurantiacus, having a            thermostability value of more than 30% determined as            Relative Activity at 80° C./70° C.;        -   optionally a pullulanase;        -   optionally a Penicillium oxalicum glucoamylase    -   ii) saccharifying using a glucoamylase enzyme;    -   iii) fermenting using a fermenting organism;    -   wherein a cellulolytic composition is present and/or added        during fermentation or simultaneous saccharification and        fermentation.        66. A process of paragraphs 1-65, comprising the steps of:    -   i) liquefying the starch-containing material at a temperature        above the initial gelatinization temperature using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion I181+G182 and optional substitution            N193F; and optionally further one of the following set of            substitutions:        -   E129V+K177L+R179E;        -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;        -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V:        -   V59A+E129V+K177L+R179E+Q254S+M284V        -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO:            1 herein for numbering);        -   optionally a protease having a thermostability value of more            than 20% determined as Relative Activity at 80° C./70° C.,            preferably derived from Pyrococcus furiosus and/or            Thermoascus aurantiacus; and        -   optionally a pullulanase;        -   optionally a Penicillium oxalicum glucoamylase shown in SEQ            ID NO: 14 having substitutions selected from the group of:        -   K79V;        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F; or        -   K79V+P11F+D26C+K33C+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or        -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or        -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for            numbering);    -   ii) saccharifying using a glucoamylase enzyme;    -   iii) fermenting using a fermenting organism;    -   wherein a cellulolytic composition is present and/or added        during fermentation or simultaneous saccharification and        fermentation.        67. A process of paragraphs 1-66, comprising the steps of:    -   i) liquefying the starch-containing material at a temperature        between 80-90° C. using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion I181+G182 and optional substitution            N193F; and optionally further one of the following set of            substitutions:        -   E129V+K177L+R179E;        -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;        -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;        -   V59A+E129V+K177L+R179E+Q254S+M284V        -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO:            1 herein for numbering).        -   optionally a protease having a thermostability value of more            than 20% determined as Relative Activity at 80° C./70° C.,            preferably derived from Pyrococcus furiosus and/or            Thermoascus aurantiacus; and        -   optionally a pullulanase;        -   optionally a Penicillium oxalicum glucoamylase shown in SEQ            ID NO: 14 having substitutions selected from the group of:        -   K79V;        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F; or        -   K79V+P11F+D26C+K33C+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or        -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or        -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for            numbering);    -   ii) saccharifying using a glucoamylase enzyme;    -   iii) fermenting using a fermenting organism;    -   wherein a cellulolytic composition is present and/or added        during fermentation or simultaneous saccharification and        fermentation.        68. A process of any of paragraphs 1-67, comprising the steps        of:    -   i) liquefying the starch-containing material at a temperature        between 80-90° C. at a pH between 5.0 and 6.5 using:        -   an alpha-amylase derived from Bacillus stearothermophilus            having a double deletion I181+G182 and optional substitution            N193F; and optionally further one of the following set of            substitutions:        -   E129V+K177L+R179E;        -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;        -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;        -   V59A+E129V+K177L+R179E+Q254S+M284V        -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO:            1 herein for numbering).        -   a protease derived from Pyrococcus furiosus, preferably the            one shown in SEQ ID NO: 13 herein or SEQ ID NO: 29 here;        -   a Penicillium oxalicum glucoamylase shown in SEQ ID NO: 14            having substitutions selected from the group of:        -   K79V;        -   K79V+P11F+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327F; or        -   K79V+P11F+D26C+K33C+T65A+Q327F; or        -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or        -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or        -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for            numbering);    -   ii) saccharifying using a glucoamylase enzyme;    -   iii) fermenting using a fermenting organism;        wherein a cellulolytic composition, such as a Trichoderma reesei        cellulolytic composition, is present and/or added during        fermentation or simultaneous saccharification and fermentation,        in particular a Trichoderma reesei cellulolytic composition        comprising one or more polypeptides selected from the group        consisting of:    -   GH61 polypeptide having cellulolytic enhancing activity,    -   beta-glucosidase;    -   Cellobiohydrolase I;    -   Cellobiohydrolase II;        or a mixture of two, three, or four thereof.        69. The process of any of paragraphs 57-68, wherein pullulanase        present and/or added during liquefaction step i) is a family        GH57 pullulanase, wherein the pullulanase preferably includes an        X47 domain as disclosed in WO 2011/087836.        70. The process of any of paragraphs 57-69, wherein the        pullulanase is derived from a strain from the genus        Thermococcus, including Thermococcus litoralis and Thermococcus        hydrothermalis or a hybrid thereof.        71. The process of any of paragraphs 57-70, wherein the        pullulanase is the truncated Thermococcus hydrothermalis        pullulanase at site X4 or a T. hydrothermalis/T. litoralis        hybrid enzyme with truncation site X4 disclosed in WO        2011/087836 or shown in SEQ ID NO: 12 herein.        72. The process of any of paragraphs 57-71, wherein the Bacillus        stearothermophilus alpha-amylase (SEQ ID NO: 1 herein) is the        mature alpha-amylase or corresponding mature alpha-amylases        having at least 80% identity, at least 90% identity, at least        95% identity at least 96% identity at least 97% identity at        least 99% identity to the SEQ ID NO: 1.        73. The process of any of paragraphs 41-72, wherein the        Pyrococcus furiosus protease (SEQ ID NO: 13 herein or SEQ ID NO:        29 herein) and/or Thermoascus aurantiacus protease (SEQ ID        NO: 3) is the mature protease or corresponding mature protease        having at least 80% identity, at least 90% identity, at least        95% identity at least 96% identity at least 97% identity at        least 99% identity to the SEQ ID NO: 13, SEQ ID NO: 29 herein,        or SEQ ID NO: 3 herein, respectively.        74. The process of any of paragraphs 48-73, wherein the        Penicillium oxalicum glucoamylase (SEQ ID NO: 14 herein) is the        mature glucoamylase or corresponding mature glucoamylase having        at least 80% identity, at least 90% identity, at least 95%        identity at least 96% identity at least 97% identity at least        99% identity to the SEQ ID NO: 14 herein.        75. The process of paragraphs 1-74, wherein the cellulolytic        composition is derived from a strain of Trichoderma, in        particular Trichoderma reesei, or a strain of Humicola, in        particular Humicola insolens, or a strain of Chrysosporium, in        particular Chrysosporium lucknowense.        76. The process of paragraphs 1-75, wherein the cellulolytic        composition comprises a beta-glucosidase, a cellobiohydrolase        and an endoglucanase.        77. The process of any of paragraphs 1-76, wherein the        cellulolytic composition comprising one or more polypeptides        selected from the group consisting of:    -   GH61 polypeptide having cellulolytic enhancing activity,    -   beta-glucosidase;    -   Cellobiohydrolase I;    -   Cellobiohydrolase II;        or a mixture of two, three, or four thereof.        78. The process of any of paragraphs 1-77, wherein the        cellulolytic composition comprises a beta-glucosidase,        preferably one derived from a strain of the genus Aspergillus,        such as Aspergillus oryzae, such as the one disclosed in WO        2002/095014 or the fusion protein having beta-glucosidase        activity disclosed in WO 2008/057637, or Aspergillus fumigatus,        such as the one disclosed in WO 2005/047499 or SEQ ID NO: 22        herein or an Aspergillus fumigatus beta-glucosidase variant        disclosed in WO 2012/044915 or a strain of the genus a strain        Penicillium, such as a strain of the Penicillium brasilianum        disclosed in WO 2007/019442, or a strain of the genus        Trichoderma, such as a strain of Trichoderma reesei.        79. The process of any one of paragraphs 1-78, wherein the        cellulolytic composition comprises a GH61 polypeptide having        cellulolytic enhancing activity such as one derived from the        genus Thermoascus, such as a strain of Thermoascus aurantiacus,        such as the one described in WO 2005/074656 as SEQ ID NO: 2; or        one derived from the genus Thielavia, such as a strain of        Thielavia terrestris, such as the one described in WO        2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8; or one derived        from a strain of Aspergillus, such as a strain of Aspergillus        fumigatus, such as the one described in WO 2010/138754 as SEQ ID        NO: 1 and SEQ ID NO: 2; or one derived from a strain derived        from Penicillium, such as a strain of Penicillium emersonii,        such as the one disclosed in WO 2011/041397 or SEQ ID NO: 23        herein.        80. The process of any one of paragraphs 1-79, wherein the        cellulolytic composition comprises a cellobiohydrolase I (CBH        I), such as one derived from a strain of the genus Aspergillus,        such as a strain of Aspergillus fumigatus, such as the Cel7a        CBHI disclosed in SEQ ID NO: 2 in WO 2011/057140 or SEQ ID NO:        24 herein, or a strain of the genus Trichoderma, such as a        strain of Trichoderma reesei.        81. The process of any one of paragraphs 1-80, wherein the        cellulolytic composition comprises a cellobiohydrolase II (CBH        II, such as one derived from a strain of the genus Aspergillus,        such as a strain of Aspergillus fumigatus; such as the one        disclosed as SEQ ID NO: 25 herein or a strain of the genus        Trichoderma, such as Trichoderma reesei, or a strain of the        genus Thielavia, such as a strain of Thielavia terrestris, such        as cellobiohydrolase II CEL6A from Thielavia terrestris.        82. The process of any one of paragraphs 1-81, wherein the        cellulolytic composition comprises a GH61 polypeptide having        cellulolytic enhancing activity and a beta-glucosidase.        83. The process of any one of paragraphs 1-82, wherein the        cellulolytic composition comprises a GH61 polypeptide having        cellulolytic enhancing activity and a beta-glucosidase.        84. The process of any one of paragraphs 1-83, wherein the        cellulolytic composition comprises a GH61 polypeptide having        cellulolytic enhancing activity, a beta-glucosidase, and a CBHI.        85. The process of any one of paragraphs 1-84, wherein the        cellulolytic composition comprises a GH61 polypeptide having        cellulolytic enhancing activity, a beta-glucosidase, a CBHI, and        a CBHII.        86. The process of any of paragraphs 1-85, wherein the        cellulolytic composition is a Trichoderma reesei cellulolytic        enzyme composition, further comprising Thermoascus aurantiacus        GH61A polypeptide having cellulolytic enhancing activity (SEQ ID        NO: 2 in WO 2005/074656), and Aspergillus oryzae        beta-glucosidase fusion protein (WO 2008/057637).        87. The process of any of paragraphs 1-86, wherein the        cellulolytic composition is a Trichoderma reesei cellulolytic        enzyme composition, further comprising Thermoascus aurantiacus        GH61A polypeptide having cellulolytic enhancing activity (SEQ ID        NO: 2 in WO 2005/074656) and Aspergillus fumigatus        beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499) or SEQ ID NO:        22 herein.        88. The process of any one of paragraphs 1-87, wherein the        cellulolytic composition is a Trichoderma reesei cellulolytic        enzyme composition further comprising Penicillium emersonii        GH61A polypeptide having cellulolytic enhancing activity        disclosed in WO 2011/041397 (SEQ ID NO: 23 herein) and        Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO        2005/047499) or SEQ ID NO: 22 herein or a variant thereof with        the following substitutions: F100D, S283G, N456E, F512Y.        89. The process of any of paragraphs 1-88, wherein the        cellulolytic composition comprises one or more of the following        components    -   (i) an Aspergillus fumigatus cellobiohydrolase I;    -   (ii) an Aspergillus fumigatus cellobiohydrolase II;    -   (iii) an Aspergillus fumigatus beta-glucosidase or variant        thereof; and    -   (iv) a Penicillium sp. GH61 polypeptide having cellulolytic        enhancing activity;    -   or homologs thereof.        90. The process of any of paragraphs 1-89, wherein the        cellulolytic composition is dosed from 0.0001-3 mg EP/g DS,        preferably 0.0005-2 mg EP/g DS, preferably 0.001-1 mg/g DS, more        preferred from 0.005-0.5 mg EP/g DS, even more preferred        0.01-0.1 mg EP/g DS.        91. An enzyme composition comprising:    -   an alpha-amylase;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.;    -   optionally a pullulanase; and    -   optionally a carbohydrate-source generating enzyme.        92 The composition of paragraph 91, wherein the alpha-amylase is        a bacterial or fungal alpha-amylase.        93. The composition of any of paragraphs 91-92, wherein the        alpha-amylase is from the genus Bacillus, such as a strain of        Bacillus stearothermophilus, in particular a variant of a        Bacillus stearothermophilus alpha-amylase, such as the one shown        in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein.        94. The composition of paragraph 93, wherein the Bacillus        stearothermophilus alpha-amylase or variant thereof is        truncated, preferably to have around 491 amino acids.        95. The composition of any of paragraphs 91-94, wherein the        Bacillus stearothermophilus alpha-amylase has a double deletion        of positions I181+G182, and optionally a N193F substitution, or        deletion of R179+G180 (using SEQ ID NO: 1 for numbering).        96. The composition of any of paragraphs 91-95, wherein the        Bacillus stearothermophilus alpha-amylase has a substitution in        position S242, preferably S242Q substitution.        97. The composition of any of paragraphs 91-96, wherein the        Bacillus stearothermophilus alpha-amylase has a substitution in        position E188, preferably E188P substitution.        98. The composition of any of paragraphs 91-97, wherein the        alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂)        of at least 10, such as at least 15, such as at least 20, such        as at least 25, such as at least 30, such as at least 40, such        as at least 50, such as at least 60, such as between 10-70, such        as between 15-70, such as between 20-70, such as between 25-70,        such as between 30-70, such as between 40-70, such as between        50-70, such as between 60-70.        99. The composition of any of paragraphs 91-98, wherein the        alpha-amylase is selected from the group of Bacillus        stearothermophilus alpha-amylase variants:    -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S:    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   I181*+G182*+N193F+V59A+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S.        100. The composition of any of paragraphs 91-99, wherein the        protease with a thermostability value of more than 25%        determined as Relative Activity at 80° C./70° C.        101. The composition of any of paragraphs 91-100, wherein the        protease has a thermostability of more than 30%, more than 40%,        more than 50%, more than 60%, more than 70%, more than 80%, more        than 90%, more than 100%, such as more than 105%, such as more        than 110%, such as more than 115%, such as more than 120%        determined as Relative Activity at 80° C./70° C.        102. The composition of any of paragraphs 91-101, wherein the        protease has a thermostability of between 20 and 50%, such as        between 20 and 40%, such as 20 and 30% determined as Relative        Activity at 80° C./70° C.        103. The composition of any of paragraphs 91-102, wherein the        protease has a thermostability between 50 and 115%, such as        between 50 and 70%, such as between 50 and 60%, such as between        100 and 120%, such as between 105 and 115% determined as        Relative Activity at 80° C./70° C.        104. The composition of any of paragraphs 91-103, wherein the        protease has a thermostability of more than 10%, such as more        than 12%, more than 14%, more than 16%, more than 18%, more than        20%, more than 30%, more than 40%, more that 50%, more than 60%,        more than 70%, more than 80%, more than 90%, more than 100%,        more than 110% determined as Relative Activity at 85° C./70° C.        105. The composition of any of paragraphs 91-10486-99, wherein        the protease has thermostability of between 10 and 50%, such as        between 10 and 30%, such as between 10 and 25% determined as        Relative Activity at 85° C./70° C.        106. The composition of any of paragraphs 91-105, wherein the        protease has a themostability above 60%, such as above 90%, such        as above 100%, such as above 110% at 85° C. as determined using        the Zein-BCA assay.        107. The composition of any of paragraphs 91-106, wherein the        protease has a themostability between 60-120, such as between        70-120%, such as between 80-120%, such as between 90-120%, such        as between 100-120%, such as 110-120% at 85° C. as determined        using the Zein-BCA assay.        108. The composition of any of paragraphs 91-107, wherein the        protease is of fungal origin.        109. The composition of any of paragraphs 91-108, wherein the        protease is a variant of the metallo protease derived from a        strain of the genus Thermoascus, preferably a strain of        Thermoascus aurantiacus, especially Thermoascus aurantiacus        CGMCC No. 0670.        110. The composition of any of paragraphs 91-109, wherein the        protease is a variant of the metallo protease disclosed as the        mature part of SEQ ID NO: 2 disclosed in WO 2003/048353 or the        mature part of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 3        herein with the following mutations:

D79L+S87P+A112P+D142L: D79L+S87P+D142L; orA27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

111. The composition of any of paragraphs 91-110, wherein the proteasevariant has at least 75% identity preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, but less than 100% identity to the mature part of thepolypeptide of SEQ ID NO: 2 disclosed in WO 2003/048353 or the maturepart of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 3 herein.112. The composition of any of paragraphs 91-111, wherein the proteasevariant of the Thermoascus aurantiacus protease shown in SEQ ID NO: 3herein is one of the following:

-   -   D79L S87P D142L;    -   D79L S87P A112P D142L;    -   D79L Y82F S87P A112P D142L;    -   S38T D79L S87P A112P A126V D142L;    -   D79L Y82F S87P A112P A126V D142L;    -   A27K D79L S87P A112P A126V D142L;    -   S49P D79L S87P A112P D142L;    -   S50P D79L S87P A112P D142L;    -   D79L S87P D104P A112P D142L;    -   D79L Y82F S87G A112P D142L;    -   S70V D79L Y82F S87G Y97W A112P D142L;    -   D79L Y82F S87G Y97W D104P A112P D142L;    -   S70V D79L Y82F S87G A112P D142L;    -   D79L Y82F S87G D104P A112P D142L;    -   D79L Y82F S87G A112P A126V D142L;    -   Y82F S87G S70V D79L D104P A112P D142L;    -   Y82F S87G D79L D104P A112P A126V D142L;    -   A27K D79L Y82F S87G D104P A112P A126V D142L.        113. The composition of any of paragraphs 91-112, wherein the        protease is of bacterial origin.        114. The composition of any of paragraphs 91-113, wherein the        protease is derived from a strain of Pyrococcus, preferably a        strain of Pyrococcus furiosus.        115. The composition of any of paragraphs 91-114, wherein the        protease is the one shown in SEQ ID NO: 1 in U.S. Pat. No.        6,358,726, SEQ ID NO: 13 herein or SEQ ID NO: 29 herein.        116. The composition of any of paragraphs 91-115, wherein the        protease is one having at least 80%, such as at least 85%, such        as at least 90%, such as at least 95%, such as at least 96%,        such as at least 97%, such as at least 98%, such as at least 99%        identity to in SEQ ID NO: 1 in U.S. Pat. No. 6,358,726, SEQ ID        NO: 13 herein or SEQ ID NO: 29 herein.        117. The composition of any of paragraphs 91-116, wherein a        carbohydrate-source generating enzyme is a glucoamylase.        118. The composition of any of paragraphs 91-117, wherein the        carbohydrate-source generating enzyme is a glucoamylase having a        heat stability at 85° C., pH 5.3, of at least 20%, such as at        least 30%, preferably at least 35%.        119. The composition of any of paragraphs 91-118, wherein the        carbohydrate-source generating enzyme is a glucoamylase having a        relative activity pH optimum at pH 5.0 of at least 90%,        preferably at least 95%, preferably at least 97%.        120. The composition of any of paragraphs 91-120, wherein the        carbohydrate-source generating enzyme is a glucoamylase having a        pH stability at pH 5.0 of at least at least 80%, at least 85%,        at least 90%.        121. The composition of any of paragraphs 91-120, wherein the        carbohydrate-source generating enzyme is a glucoamylase,        preferably derived from a strain of the genus Penicillium,        especially a strain of Penicillium oxalicum disclosed as SEQ ID        NO: 2 in WO 2011/127802 or SEQ ID NOs: 9 or 14 herein.        122. The composition of any of paragraphs 91-121, wherein the        glucoamylase has at least 80%, more preferably at least 85%,        more preferably at least 90%, more preferably at least 91%, more        preferably at least 92%, even more preferably at least 93%, most        preferably at least 94%, and even most preferably at least 95%,        such as even at least 96%, at least 97%, at least 98%, at least        99% or 100% identity to the mature polypeptide shown in SEQ ID        NO: 2 in WO 2011/127802 or SEQ ID NOs: 9 or 14 herein.        123. The composition of any of paragraphs 91-122, wherein the        carbohydrate-source generating enzyme is a variant of the        glucoamylase derived from a strain of Penicillium oxalicum        disclosed as SEQ ID NO: 2 in WO 2011/127802 having a K79V        substitution (using the mature sequence shown in SEQ ID NO: 14        for numbering).        124. The composition of any of paragraphs 91-123, further        comprising a glucoamylase.        125. The composition of any of paragraphs 91-124, further        comprising a pullulanase.        126. The composition of any of paragraphs 91-125, comprising    -   an alpha-amylase derived from Bacillus stearothermophilus;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.        derived from Pyrococcus furiosus or Thermoascus aurantiacus;    -   optionally a pullulanase;    -   optionally a glucoamylase derived from Penicillium oxalicum.        127. The composition of any of paragraphs 91-126, comprising    -   an alpha-amylase derived from Bacillus stearothermophilus;    -   a protease having a thermostability value of more than 20%        determined as Relative Activity at 80° C./70° C. derived from        Pyrococcus furiosus or Thermoascus aurantiacus;    -   optionally a pullulanase;    -   a glucoamylase derived from Penicillium oxalicum.        128. The composition of any of paragraphs 91-127, comprising    -   an alpha-amylase, preferably derived from Bacillus        stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂ of at least 10;    -   optionally a protease, preferably derived from Pyrococcus        furiosus or Thermoascus aurantiacus, having a thermostability        value of more than 20% determined as Relative Activity at 80°        C./70° C.;    -   optionally a pullulanase;    -   optionally a glucoamylase derived from Penicillium oxalicum.        129. The composition of any of paragraphs 91-128, comprising    -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182 and substitution N193F; and        optionally further one of the following set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   V59A+E129V+K177L+R179E+Q254S+M284V;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering);    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.        derived from Pyrococcus furiosus and/or Thermoascus aurantiacus;    -   optionally a pullulanase;    -   optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 14        having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering).        130. The composition of any of paragraphs 91-129 comprises:    -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion I181+G182+N193F; and further one of the        following set of substitutions:    -   E129V+K177L+R179E;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+Q254S+M284V;    -   V59A+E129V+K177L+R179E+Q254S+M284V    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1        herein for numbering).    -   a protease derived from Pyrococcus furiosus, preferably the one        shown in SEQ ID NO: 13 herein or SEQ ID NO: 29 herein;    -   a Penicillium oxalicum glucoamylase shown in SEQ ID NO: 14        having substitutions selected from the group of:    -   K79V;    -   K79V+P11F+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327F; or    -   K79V+P11F+D26C+K33C+T65A+Q327F; or    -   K79V+P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; or    -   K79V+P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; or    -   K79V+P11F+T65A+Q327W+E501V+Y504T (using SEQ ID NO: 14 for        numbering);        131. The compositions of any of paragraphs 91-130, comprising    -   an alpha-amylase derived from Bacillus stearothermophilus having        a double deletion        I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using        SEQ ID NO: 1 herein for numbering).    -   a protease derived from Pyrococcus furiosus preferably the one        shown in SEQ ID NO: 13 herein or SEQ ID NO: 29 herein;    -   a Penicillium oxalicum glucoamylase shown in SEQ ID NO: 14        having substitutions selected from the group of:    -   K79V+P11F+T65A+Q327F    -   K79V+P2N+P4F+P11F+T65A+Q327F (using SEQ ID NO: 14 for        numbering).        132. The composition of any of paragraphs 126-131, wherein the        pullulanase is a family GH57 pullulanase, wherein the        pullulanase preferably includes an X47 domain as disclosed in WO        2011/087836.        133. The composition of any of paragraphs 126-132, wherein the        pullulanase is derived from a strain from the genus        Thermococcus, including Thermococcus litoralis and Thermococcus        hydrothermalis or a hybrid thereof.        134. The composition of any of paragraphs 126-133, wherein the        pullulanase is the truncated Thermococcus hydrothermalis        pullulanase at site X4 or a T. hydrothermalis/T. litoralis        hybrid enzyme with truncation site X4 disclosed in WO        2011/087836 or shown in SEQ ID NO: 12 herein.        135. The composition of any of paragraphs 126-134, wherein the        Bacillus stearothermophilus alpha-amylase (SEQ ID NO: 1 herein),        or a variant thereof, is the mature alpha-amylase or        corresponding mature alpha-amylases having at least 80%        identity, at least 90% identity, at least 95% identity at least        96% identity at least 97% identity at least 99% identity to SEQ        ID NO: 1.        136. The composition of any of paragraphs 91-135, wherein the        Pyrococcus furiosus protease (SEQ ID NO: 13 herein or SEQ ID NO:        29 herein) and/or Thermoascus aurantiacus protease (SEQ ID NO: 3        herein), or a variant thereof, is the mature protease or        corresponding mature protease having at least 80% identity, at        least 90% identity, at least 95% identity at least 96% identity        at least 97% identity at least 99% identity to SEQ ID NO: 13        herein or SEQ ID NO: 29 herein, or SEQ ID NO: 3, respectively.        137. The composition of any of paragraphs 91-136, wherein the        Penicillium oxalicum glucoamylase (SEQ ID NO: 14 herein), or a        variant thereof, is the mature glucoamylase or corresponding        mature glucoamylase having at least 80% identity, at least 90%        identity, at least 95% identity at least 96% identity at least        97% identity at least 99% identity to the SEQ ID NO: 14 herein.

1. A process for producing fermentation products from starch-containingmaterial comprising the steps of: i) liquefying the starch-containingmaterial at a temperature above the initial gelatinization temperatureusing: an alpha-amylase; optionally a protease having a thermostabilityvalue of more than 20% determined as Relative Activity at 80° C./70° C.;and optionally a carbohydrate-source generating enzyme; ii)saccharifying using a carbohydrate-source generating enzyme; iii)fermenting using a fermenting organism; wherein a cellulolyticcomposition is present or added during fermentation or simultaneoussaccharification and fermentation.
 2. The process of claim 1, whereinthe pH during liquefaction is between above 5.0-6.5, such as above5.0-6.0, such as above 5.0-5.5, such as between 5.2-6.2, such as around5.2, such as around 5.4, such as around 5.6, such as around 5.8.
 3. Theprocess of claim 1, wherein the fermentation product is an alcohol,preferably ethanol, especially fuel ethanol, potable ethanol and/orindustrial ethanol.
 4. The process of claim 1, wherein the alpha-amylaseis from the genus Bacillus, such as a strain of Bacillusstearothermophilus, in particular a variant of a Bacillusstearothermophilus alpha-amylase, such as the one shown in SEQ ID NO: 1herein.
 5. The process of claim 4, wherein the Bacillusstearothermophilus alpha-amylase has a double deletion of positionsI181+G182 and optionally a N193F substitution, or deletion of R179+G180(using SEQ ID NO: 1 for numbering).
 6. The process of claim 1, whereinthe protease is a variant of the metallo protease derived from a strainof the genus Thermoascus, preferably a strain of Thermoascusaurantiacus, especially Thermoascus aurantiacus CGMCC No.
 0670. 7. Theprocess of claim 1, wherein the protease is derived from a strain ofPyrococcus, preferably a strain of Pyrococcus furiosus.
 8. The processof claim 1, wherein the carbohydrate-source generating enzyme presentand/or added during liquefaction step i) is a glucoamylase, preferablyderived from a strain of the genus Penicillium, especially a strain ofPenicillium oxalicum disclosed as SEQ ID NOs: 9 or 14 herein.
 9. Theprocess of claim 1, further wherein a glucoamylase is present and/oradded during saccharification and/or fermentation.
 10. The process ofclaim 1, wherein the glucoamylase present and/or added duringsaccharification and/or fermentation is of fungal origin, preferablyfrom a stain of Aspergillus, preferably A. niger, A. awamori, or A.oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain ofTalaromyces, preferably Talaromyces emersonii, or a strain ofPycnoporus, or a strain of Gloeophyllum, such as a strain ofGloeophyllum sepiarium or Gloeophyllum trabeum or a strain of theNigrofomes.
 11. The process of claim 1, comprising the steps of: i)liquefying the starch-containing material at a temperature above theinitial gelatinization temperature using: an alpha-amylase derived fromBacillus stearothermophilus; optionally a protease having athermostability value of more than 20% determined as Relative Activityat 80° C./70° C., preferably derived from Pyrococcus furiosus and/orThermoascus aurantiacus; and a Penicillium oxalicum glucoamylase; ii)saccharifying using a glucoamylase enzyme; iii) fermenting using afermenting organism; wherein a cellulolytic composition is present oradded during fermentation or simultaneous saccharification andfermentation.
 12. The process of claim 1, wherein the cellulolyticcomposition is derived from a strain of Trichoderma, in particularTrichoderma reesei, or a strain of Humicola, in particular Humicolainsolens, or a strain of Chrysosporium, in particular Chrysosporiumlucknowense.
 13. The process of claim 1, wherein the cellulolyticcomposition is a Trichoderma reesei cellulolytic enzyme compositionfurther comprising Penicillium emersonii GH61A polypeptide havingcellulolytic enhancing activity disclosed in SEQ ID NO: 23 herein andAspergillus fumigatus beta-glucosidase disclosed in SEQ ID NO: 22 hereinor a variant thereof with the following substitutions: F100D, S283G,N456E, F512Y.
 14. An enzyme composition comprising: an alpha-amylase;optionally a protease having a thermostability value of more than 20%determined as Relative Activity at 80° C./70° C.; optionally apullulanase; and a carbohydrate-source generating enzyme.
 15. Thecomposition of claim 14, wherein the alpha-amylase is from the genusBacillus, such as a strain of Bacillus stearothermophilus, in particulara variant of a Bacillus stearothermophilus alpha-amylase, such as theone shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein. 16.The composition of claim 14, wherein the Bacillus stearothermophilusalpha-amylase has a double deletion of positions I181+G182, andoptionally a N193F substitution, or deletion of R179+G180 (using SEQ IDNO: 1 for numbering).
 17. The composition of claim 14, wherein theprotease is a variant of the metallo protease derived from a strain ofthe genus Thermoascus, preferably a strain of Thermoascus aurantiacus,especially Thermoascus aurantiacus CGMCC No.
 0670. 18. The compositionof claim 14, wherein the protease is derived from a strain ofPyrococcus, preferably a strain of Pyrococcus furiosus.
 19. Thecomposition of claim 14, wherein the carbohydrate-source generatingenzyme is a glucoamylase having a heat stability at 85° C., pH 5.3, ofat least 20%, such as at least 30%, preferably at least 35%.
 20. Thecomposition of claim 14, wherein the carbohydrate-source generatingenzyme is a glucoamylase, preferably derived from a strain of the genusPenicillium, especially a strain of Penicillium oxalicum disclosed asSEQ ID NOs: 9 or 14 herein.